Progress report. Sterol absorption-from lumen to liver. polymolecular aggregates of bile salts, monoglycerides, fatty acids,

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1 Progress report Gut, 1971, 12, Sterol absorption-from lumen to liver Sterols are compounds of great biological importance which are found in animals and plants'. They are insoluble in water and are chemically rather unreactive and these qualities are appropriate to their role as structural components of cellular membranes. Cholesterol is the predominant mammalian sterol but biosynthetic precursors such as lathosterol and 7- dehydrocholesterol are present in significant amounts in the intestinal mucosa, skin, and other tissues. The major plant sterols are,b-sitosterol, campesterol, and stigmasterol. Sterols are present in large amounts in mammalian diets. The content of individual sterols in a diet depends, among other things, on the relative proportions of foodstuffs of plant and animal origin. Following ingestion exogenous sterol is mixed in stomach and small intestine with endogenous sterol; the latter is mainly cholesterol which is added in bile and shed epithelial cells but in some animals endogenous sterols are also ingested during grooming of the skin2. The bulk of dietary sterol is present as the free alcohol but a variable proportion of both plant and animal sterols is present in the form of steryl esters which are hydrolysed in the gut by the pancreatic enzyme cholesterol esterase3. The contents of the small intestine can normally be separated by ultracentrifugation into three phases: sediment, supranatant emulsion, and an aqueous micellar phase. The mixed micelles of the last phase are watersoluble polymolecular aggregates of bile salts, monoglycerides, fatty acids, and phospholipids; they are capable of dissolving water-insoluble organic compounds such as sterols within their hydrocarbon core4. It is usually stated that the sterols in the small intestinal lumen are distributed between the emulsion and micellar phases5 but this conclusion was reached on the basis of experiments in which large amounts of triglyceride were fed. In rats fed a normal laboratory diet little evidence of a significant emulsion was found and the bulk of intraluminal sterol was found in the sediment6. Sterols can also be found in the small intestinal sediment in man7 and it is likely that the importance of the emulsion phase in sterol absorption has been overemphasized. At varying levels of the rat small intestine the micellar sterol pattern was found to be similar to that of the sediment6. This suggests that different sterols have a similar solubility in micelles and also that sediment sterol is available for absorption. As micellar solution is obligatory for mucosal uptake of sterols3 absorption can take place only from the small intestine and it is now clear that it is the upper small intestine which is quantitatively most important. In this area hydrolysis of triglycerides provides relatively large amounts of monoglycerides and fatty acids for incorporation into mixed micelles; this increases the amount of sterol which can be held in solution and thsws the amount available for absorption. Less monoglyceride and fatty acids are generated 411

2 412 Neil McIntyre lower in the intestine and sterol solubility in micelles is correspondingly decreased. The mode of transfer of sterols from micelles into mucosal cells is not known. It appears likely that it results from molecular collision and not by incorporation of the whole micelle into the surface membrane of the epithelium. Only free sterols are taken up into the mucosa. No absorption occurs when esters resistant to the action of cholesterol esterase are administered3 8. There is evidence that mucosal cells can also discriminate in their uptake of individual free sterols. Campesterol enters the mucosa more easily than fl-sitosterol6'9 and both of these plant sterols are absorbed poorly compared with cholesterol, a finding due, at least in part, to differences in uptake from the lumen10. The nature of this discriminatory process has not yet been elucidated but there is evidence to suggest that it depends on the metabolic integrity of the intestinal mucosall. The precise mechanisms involved in sterol transport across the epithelial cells are also poorly understood. Labelled cholesterol and other sterols taken up from the lumen appear to become distributed uniformly among the subcellular fractions of the mucosal cell when conventional techniques are employed'2 3"4. One group of workers has claimed that there is no increase in the cholesterol content of mucosal cells during cholesterol absorption'5; from this they argued that the process of absorption must be a displacement of existing sterol from the lipoproteins of the cell. However other workers have found a pronounced rise in the concentration of mucosal sterol with cholesterol feeding,"' and it seems equally likely that the mass of sterol in mucosal cells may be an important determinant of the rate of cholesterol absorption. When absorbed sterols leave the mucosa they enter intestinal lymph. Following a cholesterol-free fatty meal the cholesterol content of lymph chylomicrons obtained from experimental animals is low and most of it is present in the unesterified form'7. As the cholesterol content of the diet is increased the concentration of lymph cholesterol also increases and the proportion present in the esterified form increases to around 60%. Studies with labelled cholesterol have revealed that a larger proportion (70-80%) of luminal cholesterol is esterified before entry into the lymph than would appear to be the case from straightforward chemical estimations. This discrepancy is attributable to the dilution of the lymphatic free cholesterol with cholesterol of endogenous origin'8. The increase in the amount of total lymph ester with cholesterol feeding suggests that esterification may be an important factor in determining the rate of cholesterol absorption. However, when the rate of cholesterol absorption was varied by feeding different amounts of 14C-cholesterol and by the addition to the diet of cholesterol analogues, there was no change in the relative proportions of lymphatic '4C-cholesterol in free and esterified forms. Furthermore epicholesterol, a structural analogue of cholesterol, is well absorbed and transported into lymph even though it is not esterified by the mucosa20. Thus a precise assessment of the quantitative importance of esterification in sterol absorption is not yet possible. Esterification in the small intestinal wall is believed to result from the action of mucosal cholesterol esterase. This enzyme appears to have similar properties to pancreatic cholesterol esterase; it is capable of both hydrolysis

3 Sterol absorption-from lumen to liver and synthesis of sterol esters and bile salts are necessary cofactors for activity3. Following diversion of pancreatic juice it has been claimed that mucosal cholesterol esterase is greatly reduced and that cholesterol absorption is impaired21. On this basis it has been suggested that the pancreas is the source of the mucosal enzyme. However, others believe that a separate mucosal enzyme, synthesized locally, is responsible for mucosal production of cholesterol ester. Prolonged feeding of cholesterol was found to result in an increase in the cholesterol esterifying activity of the intestinal mucosa without corresponding change in its hydrolytic activity or in the activity of pancreatic cholesterol esterase22. The pancreas may also have other functions in cholesterol absorption, as a heat-stable factor has been found in pancreatic juice which stimulates cholesterol esterification23. The evidence on the role of the pancreas in the mucosal process of sterol absorption is contradictory and further work is needed in this field. Cholesterol of intestinal origin is carried in lymph in triglyceride-rich lipoprotein particles. The role of the large chylomicrons (Sf > 400) is well established as most studies of lymph cholesterol have been done either on whole lymph or on the chylomicron fraction'7. Chylomicrons are large particles composed of a lipid core and a surface membrane which can be separated by a variety of techniques. The core contains 99 % of the triglyceride of the chylomicron, all of the cholesterol ester, a fraction of the free cholesterol, but none of the phospholipid; by contrast the membrane is composed of phospholipid, free cholesterol, and protein'7. In the rat, the smaller very low density lipoproteins (Sf ) are also carriers of absorbed cholesterol and the distribution of cholesterol between chylomicrons and very low density lipoproteins may depend on the fatty acid composition of the dietary triglycerides The structure of very low density lipoprotein particles has not yet been clarified. Absorbed cholesterol is carried via the thoracic duct to the systemic circulation and % of chylomicron cholesterol is rapidly taken up by the liver'8. Chylomicron triglyceride, however, is largely assimilated in extrahepatic tissues following its hydrolysis by lipoprotein lipase26. This discrepancy in the fate of different chylomicron lipids has recently been investigated27. When chylomicrons were injected into hepatectomized rats triglyceride was rapidly removed from the circulation while cholesteryl ester accumulated in the plasma in particulate remnants. These contained approximately 13 % of cholesteryl ester by weight and when they were injected into intact animals the liver removed their cholesteryl ester much more rapidly than that of injected chylomicrons. This rapid removal was not due simply to the high content of cholesteryl ester as very low density lipoprotein cholesterol is removed more slowly from the plasma than chylomicron cholesterol25 even though the concentration of cholesterol is appreciably higher in very low density lipoproteins. One consequence of excessive absorption of cholesterol is the accumulation by the liver of large amounts of cholesteryl ester'8. This ester is not the same as that transported from the intestine. Although the hepatic uptake of lymph cholesteryl ester occurs without hydrolysis a slow cleavage follows and the resulting free sterol exchanges with the free cholesterol of the rest of the liver, the plasma, and the remainder of the exchangeable pool. Reesterification also occurs and it is this process which results in cholesterol ester accumulation in the liver. 413

4 414 Neil McIntyre The role of bile salts in the overall process of cholesterol absorption cannot be overemphasized. When bile is diverted from the intestine there is neither mucosal uptake of cholesterol nor transfer into lymph. The bile salts perform several functions. Their most important property is probably their key role in micelle formation. Not only are they important constituents of micelles4 but by enhancing the activity of pancreatic lipase28 they also help to provide the monoglycerides and fatty acids which expand the micelles and thus increase their capacity for solubilizing sterols. In addition bile salts are essential for intraluminal hydrolysis of ingested cholesteryl esters; all bile salts aid in solubilizing the substrate and trihydroxy bile salts protect pancreatic cholesterol esterase from proteolytic inactivation and act as cofactors for hydrolytic activity3. At a mucosal level bile salts probably activate cellular esterification of cholesterol, and recent work suggests that they might also play an important role in the transfer of cholesterol from the intestinal wall to lymph29. It is now well established that in man a high plasma cholesterol level is associated with an increased incidence of atheroma and coronary artery disease. It is therefore hardly surprising that there is a search for methods of diminishing cholesterol absorption in the hope that plasma cholesterol levels would also be reduced. In the preceding account of cholesterol absorption a composite picture has been drawn of the physiological mechanisms which are responsible for the transport of cholesterol from the intestinal lumen to the liver, and from this it should be possible to predict in a logical fashion ways in which cholesterol absorption could be either enhanced or impaired. It should be pointed out, however, that this picture has been drawn from a large number of human and animal studies and little comment has been made about the type of animal investigated. Species variation is important in the study of cholesterol absorption and this should be borne in mind when predicting the likely effectiveness of methods for the reduction of cholesterol absorption in man. At first sight it would appear that the easiest method of modifying cholesterol absorption would be to change the amount of cholesterol ingested. In the rabbit, rat, and other animals the absolute amount of cholesterol absorbed does increase with increasing dosage'. It is generally believed that with an increased intake the percentage absorption falls but recently it was claimed that in the rat the proportion of cholesterol transferred into lymph was constant over a wide range of administered cholesterol30. Several studies suggest that man has a limited capacity to absorb cholesterol as absorption does not appear to increase with increase in dietary intake31'32 but this view has been challenged recently33. Varying the dose of cholesterol over wide limits had little effect on plasma cholesterol when the composition of the diet was otherwise kept constant34, and it would appear that reduction of dietary cholesterol per se is an ineffective method of reducing plasma levels. Alteration of the dietary triglyceride content is an important method of influencing cholesterol absorption and in animals it is well established that feeding of either triglyceride or fatty acids increases the amount of cholesterol appearing in the lymph3. In a recent study in man cholesterol absorption was studied in several subjects with a thoracic duct fistula35. The oral administration of fat led to a striking increase in lymphatic cholesterol but the addition of cholesterol to the fat meal caused no further rise. This was interpreted as evidence that in man a large endogenous pool of cholesterol

5 Sterol absorption-from lumen to liver 415 is normally available for absorption and that the rate limiting factor is the amount of triglyceride available either for the formation of micelles or for the production of lymph lipoproteins. Serum cholesterol levels can be reduced by feeding diets rich in polyunsaturated fatty acids but although lower lymph cholesterol levels were found in dogs fed corn oil (unsaturated) than in dogs fed butter fat (saturated)36 it is not generally believed that the hypocholesterolaemic effect is related to effects on cholesterol absorption. Plant sterols have a hypocholesterolaemic action in man37 and they inhibit transfer of absorbed cholesterol into lymph in the experimental animal38. Very large doses may prevent adequate solution of cholesterol in micelles as the micellar sterol pattern reflects the relative proportions of individual sterols present in the lumen; this would impair uptake from the lumen but plant sterols may also interfere with transport and esterification of cholesterol at a mucosal level. It has been reported that plant sterols may have a hypocholesterolaemic effect unrelated to their effects on cholesterol absorption39 but it is unlikely that this would be of much practical importance as plant sterols are poorly absorbed from the intestine. More recently there have been attempts to interfere with cholesterol absorption by disruption of micelles. Two compounds have been used: cholestyramine, an anion binding resin which binds bile acids40, and neomycin, a polybasic substance which interacts primarily with fatty acids41. These substances prevent effective micelle formation and lead to a precipitation of sterols held in micellar solution. Both agents have been found to lower plasma cholesterol levels in man42,43. Both compounds also increase the faecal loss of bile acids but cholestyramine causes the greater loss as the binding of bile acids interferes with their reabsorption in the terminal ileum. Bile acids are formed from cholesterol and, although hepatic cholesterol synthesis rises in response to the stimulus of bile acid depletion44, it is likely that utilization of preformed plasma and tissue cholesterol for bile acid synthesis is an important factor in the fall of plasma cholesterol. Few other drugs have been found to block cholesterol absorption effectively but studies with cholestane-3p, S5x, 6/3-triol-have shown that it reduces cholesterol absorption in rats by 30 to 50 %45. The precise mechanism is unknown but it appears to act at a mucosal level. There is little or no information on its use in man but clearly such an agent has great therapeutic potential. Although our knowledge of the physiology of sterol absorption is expanding rapidly a major gap will remain until we know more about the intramucosal processes. When this area is clarified we will be in a better position to understand and control the absorption of a physiologically important but potentially lethal substance. References NEIL MCINTYRE Department of Medicine, Royal Free Hospital, London NW3 "Cook, R. P. (1958). Cholesterol. Chemistry, Biochemistry and Pathology. Academic Press, New York. 2Miettinen, T. Personal communication. Quoted by Gustafsson, B. E., Gustafsson, J. A., and Sjovall, J. (1966). Intestinal and fecal sterols in germ free and conventional rats. Acta chem. scand., 20, Treadwell, C. R., and Vahouny, G. V. (1968). Cholesterol absorption. In Handbook of Physiology, sect. 6, vol. 3, edited by C. F. Code and W. Heidal, pp American Physiological Society, Washington, D.C.

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