Absorption and Metabolism of Lipid in Humans

Patrick Tso and Stuart W. Weidman

Departments of Physiology and Medicine, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163

DIETARY LIPIDS

Dietary lipids can provide as much as 40% of the daily caloric intake in the Western diet. The daily dietary intake of lipid by humans in the Western world ranges between 60 and 100 g (1,2). Triglyceride (TG) is the major dietary fat in humans. Long-chain fatty acids such as the oleate (18:1) and palmitate (16:0) are the major fatty acids (FA) present. In most infant diets, fat becomes a major energy source. In human milk and in human formulas, 40% to 50% of the total calo- ries are present as fat (3). The human small intestine is presented daily with other lipids such as phospholipid (PL) and cholesterol and other sterols. Both PL and cholesterol are major constituents of bile. In humans, the biliary PL is a major contributor of the luminal PL. It has been calculated that 11 to 12 g of biliary PL enters the small intestinal lumen daily, whereas the dietary contribution is 1 to 2 g (4). The predominant sterol in the Western diet is cholesterol. However, plant sterols account for 20% to 25% of total dietary sterol (5-7). It is beyond the scope of this review to discuss the absorption of lipid soluble vitamins, and the interested readers should refer to the excellent review by Barrowman (8).

INTRALUMINAL DIGESTION OF LIPIDS

Although the majority of the digestion of TG occurs in the small intestine, the digestion starts in the stomach. Lipase activity has been reported to be present in the human gastric juice (9). This lingual lipase is derived from a group of serous glands (Von Ebner) beneath the circumvallate papillae (3). The lingual lipase plays a particularly important role in the digestion of milk TG, and this compensates for the low pancreatic activity in the newborn. Readers interested in the subject of lingual lipase should refer to the review by Hamosh (3). Lipid emulsion enters the small intestinal lumen as fine lipid droplets less than 0.5 (i-m in diameter (10,11). The combined action of the bile and the pancreatic juice brings about a marked 2 UPID ABSORPTION AND METABOUSM IN HUMANS change in the chemical and physical form of the ingested lipid emulsion. The digestion of TG is brought about by the pancreatic lipase in the upper part of the intestinal lumen. The pancreatic lipase works at the interface between the oil and the aqueous phases. The velocity of lipolysis depends on factors modifying the physicochemical properties of the interface as well as the surface area (12-15). Pancreatic lipase acts predominantly at the 1- and 3-ester bonds of TG to release 2-monoglyceride (MG) and free FA (13,16-18). Through isomerization, the 2-MG can be converted to 1-MG. However, this reaction probably occurs slowly in the intestine (19). A pancreatic esterase has been demonstrated by Hofmann and Borg- strom (20) that catalyzes the hydrolysis of 1- or 2-MG to form glycerol and FA. The pancreatic esterase works more efficiently with the 1-isomer than with the 2- isomer. In vitro studies using purified pancreatic lipase have demonstrated a potent inhibitory effect of bile salts on lipolysis of TG at concentration above critical micellar concentration (21,22). The inhibitory effect of bile salt is physiological as the concentration of bile salts in the duodenum is normally higher than the concentration of bile salt needed to observe the inhibitory effect. The pancreas synthesizes another protein that antagonizes the inhibitory action of bile salts. This factor was first isolated by Morgan et al. (23) from rat pancreatic juice. The structure and action of colipase have predominantly been elucidated by the work of Desnuelle and his group in Marseille, and Borgstrom and co-workers in Lund (22,24,25). Colipase acts by attaching to the ester bond region of the TG molecule. In turn, the lipase binds strongly to the colipase by electrostatic interactions, thereby allow- ing the hydrolysis of the TG by the lipase molecule (26). Colipase is secreted as a procolipase and is converted to the active form through trypsin digestion (27). The majority of luminal PL is phosphatidylcholine (PC). Both biliary and dietary PC are hydrolyzed in the presence of pancreatic phospholipase A2 to form lysophosphatidylcholine (LPC) and FA (28,29). Although it is generally accepted that bile PC or dietary PC is only absorbed after hydrolysis by pancreatic phospholipase A2 to form 1-lysophosphatidylcholine (28,30,31), it has been proposed that biliary PC is resistant to the action of phospholipase A2, there being an enterohe- patic circulation of bile PC (32-34). This hypothesis needs to be further investi- gated. Cholesterol ester entering the small intestinal lumen is hydrolyzed in the presence of the pancreatic cholesterol esterase to form free sterol prior to its absorption (35,36).

UPTAKE OF DIGESTED FAT BY THE ENTEROCYTE

The lipolytic products distribute themselves between the aqueous and the oil phases. In the aqueous phase, the lipolytic products exist mainly as part of the mixed bile salt micelle, although some exist in very low concentrations as monomolecular species in classical solution. This complex problem has been ably reviewed by Hofmann (37). As digestion progresses, the oil phase gets smaller in volume because of the lipolytic products passing into the mixed micelles and into LIP1D ABSORPTION AND METABOLISM IN HUMANS 3 the absorptive cells. This concept has recently been challenged by the findings of Carey and associates (38-40). They demonstrated the presence of two dispersed phases within the aqueous phase, a phase of micelles saturated with lipids and cholesterol (hydrodynamic radii, 200 A) and a phase of unilamellar vesicles (lipo- somes) with radii of 400 to 600 A saturated with bile salts (11). The unilamellar vesicles of mixed lipids saturated with bile salts may play an important role in the uptake of lipid in bile-salt deficient patients (11,41). Further experiments are needed to elucidate the physiological importance of Carey and co-workers' obser- vation. There is a consensus that the uptake of lipid digestion products is passive (42,43). Micellar solubilization increases uptake of the lipid digestion products by increasing their aqueous concentration gradient across the unstirred water layer (42,44-46). How the unstirred water layer affects lipid uptake is complex. The subject has been thoroughly reviewed by Thomson and Dietschy (43). The lipid digestion products enter the enterocytes as monomers (44,47). There are still a few interesting observations for which we do not have an explanation. First, why is p- sitosterol so poorly absorbed relative to cholesterol? The uptake of cholesterol by the enterocytes is different from that of the other lipid digestion products. The absorption of cholesterol seems to involve the collision of the micelle with the brush border membrane, and the disruption of the micelle as a result of monoglyceride and FA absorption might enhance the entry of cholesterol into the lipid membrane (48). In in vivo experiments, the cholesterol was absorbed much more efficiently than the p-sitosterol. However, this selectivity was lost in in vitro experiments. Thus, this membrane selectivity may be energy dependent. Sylven (49), by oc- cluding the blood supply to the jejunum, markedly reduced sterol uptake in the otherwise intact animal, thus supporting the theory that this membrane discrimina- tion of sterol absorption is energy dependent. Secondly, is the unstirred water layer physiologically very important? If so, how are patients with bile salt deficiency able to absorb lipid so well?

METABOLISM OF ABSORBED LIPID DIGESTION PRODUCTS

Once the lipid digestion products enter the cell, the major site of their metabolism is at the endoplasmic reticulum (50,51). The mode of transport of these digestion products is presumably by simple diffusion. A fatty acid binding protein (FABP) has been described and characterized (52,53). Ockner and Manning (53) suggested a possible role of FABP in regulating TG biosynthesis by adjusting the amount of FA made available for activation and incorporation into TG. The absorbed 2-MG and FA are reconstituted into TG through the monoglyceride pathway. The enzymes involved exist as an enzyme complex called the "triglyceride synthetase" (54). The other pathway for the synthesis of TG from the absorbed FA is the a-glycerophosphate pathway. The a-glycerophosphate pathway is particularly important under conditions where the supply of FA is far more 4 UPID ABSORPTION AND METABOLISM IN HUMANS abundant than the MG. When MG and FA are absorbed together, the MG pathway plays a more important role than the a-glycerophosphate pathway. This is supported by data both in animals (17,55) and also in humans (56). The reasons why the MG pathway is the preferred pathway are three-fold. First, the MG pathway has a Km of approximately one-hundredth that of the a-glycerophosphate pathway (57), and, thus, the rate is much faster with the former. Second, it has been suggested that the MG pathway may be preferentially activated by bile salts (58). Third, the presence of MG inhibits the a-glycerophosphate pathway and thus fa- vors the MG pathway (59). While TG is the major product of the two pathways, the diglyceride (DG) and the TG originated from these pathways seem to enter different metabolic pools. For example, only the DG from the a-glycerophosphate is used in the biosynthesis of PC with the Kennedy pathway (60). Furthermore, Mansbach and Parthasarathy (61) provided evidence that the intracellular TG pool that derives its glyceride-glycerol from luminal TG is rapidly transported into lymph as chylomicrons. In contrast, the TG pool that derives its glyceride-glycerol largely from endogenous sources is slowly transported into lymph as lipoproteins. The absorbed LPC is partially reacylated by acyl-coenzyme A (acyl-CoA) to form PC (30,62,63). A considerable portion of the absorbed LPC is hydrolyzed by lysolecithin to glycerylphosphorylcholine (GPC) and FA (64,65). The GPC released can be transported in the blood, and the FA can be used for the synthesis of TG. Cholesterol absorbed by the enterocytes enters a free cholesterol pool. This free cholesterol pool also contains cholesterol from endogenous sources. The endogenous cholesterol is derived both from nondietary cholesterol absorbed from the lumen and cholesterol synthesized de novo (66). Recent evidence, however, sup- ports the concept that there are separate cholesterol pools in the enterocytes and that the newly synthesized cholesterol is predominantly incorporated into cell membranes (67-69). Both endogenous and dietary cholesterol are transported in chylomicrons (CM) and very low density lipoproteins (VLDL) as free and esteri- fied cholesterol. It has not been firmly established whether the esterification of cholesterol is the rate limiting step for the lymphatic transport of cholesterol. Two enzymes have been reported to be responsible for the esterification of the cholesterol prior to their transport into the lymphatics. One of the enzymes is the cholesteryl ester hydrolase (also called cholesterol esterase), an enzyme of pancreatic origin. It has been reasonably well established that this mucosal enzyme, cholesteryl ester hydrolase, is derived from the pancreatic cholesterol esterase (70-72). Using immunocytochemistry, Gallo et al. (72) provided evidence that the intestinal mucosal "cholesterol esterase" is pancreatic in origin and that there exists a gradient of this enzyme along the small intestine, with the highest concentration in the duodenum. There is another enzyme present in the intestinal mucosa that is capable of esterifying the cholesterol with acyl-CoA. The enzyme is called the acyl- CoAxholesterol acyltransferase (ACAT) and is made by the enterocytes. Several lines of evidence have indicated that this enzyme may play a more important role than the cholesterol esterase in the mucosal esterification of cholesterol. First, Watt UP1D ABSORPTION AND METABOLISM IN HUMANS 5 and Simmonds (73) failed to observe an effect of pancreatic juice on the absorption and transport of luminal cholesterol. Second, using an ACAT inhibitor Heider et al. (74) provided evidence supporting the claim by Norum et al. (75) that mucosal ACAT plays an important role in the esterification and lymphatic transport of cholesterol. Third, Norum et al. (76) found that the ACAT activity was highest in the villus cells and that the activity was responsive to fasting and feeding and also to the nature of the diet. It is important to emphasize that this question of which enzyme plays a more important role in the esterification of cholesterol is not yet resolved and deserves future research. As we pointed out earlier, there is also plant sterol present in human diet. We do not understand why only approximately 5% of dietary intake of p-sitosterol is absorbed in humans (6,77-79). In patients with |3-sitosterolemia, this discrimina- tion between cholesterol and 3-sitosterol becomes less effective (80). The absorption of cholesterol is obviously of clinical importance, in particular to the genesis of atherosclerosis and vascular disease. Yet we still have a poor understanding of the process and the factors regulating this process. This subject has been reviewed by Mclntyre (48). Studies carried out by Borgstrom (78) using fecal analyses after a single meal of radioactive cholesterol was given together with labeled (3-sitosterol as a nonabsorbable marker demonstrated that cholesterol absorption increased as the dose of cholesterol administered increased from 150 mg to 2 g. This was supported by other studies carried out in humans (81-83). Kudchodkar et al. (83) also calculated from their study that both endogenous and exogenous cholesterol behaved similarly. However, a study by Borgstrom et al. (84) in thoracic lymph fistula in humans observed an increase in lymph cholesterol after cholesterol feeding. Nevertheless, a constant amount of cholesterol was transported over a range of doses of cholesterol fed. These authors interpreted this dis- crepancy as being caused by the change in the specific activity of luminal cholesterol as more and more exogenous radioactive cholesterol was added. It is well established that the level of cholesterol in plasma does not increase proportionately with the dose of dietary cholesterol fed. In humans, only moderate increase in plasma cholesterol is observed with large doses of dietary cholesterol fed (85-87).

PACKAGING OF INTESTINAL LIPOPROTEINS

The complex lipids are processed in the endoplasmic reticulum and are then sta- bilized by the protein and phospholipid coat. The apolipoproteins synthesized by the human small intestine are apo A-I (88), apo A-II (89), apo A-IV (90,91), and apo B (92). We do not yet know the sequence of the constitution of the lipid- protein droplets in the endoplasmic reticulum. Using [14C]oleate, [3H]leucine, and [14C]glucosamine, Kessler and co-workers (93) found that the lipid, protein, and sugar of the maturing CM particles are synthesized in the smooth endoplasmic reticulum, the rough endoplasmic reticulum, and the Golgi apparatus, respectively. It is important to stress that although one particular event may be processed pre- 6 UPID ABSORPTION AND METABOLISM IN HUMANS dominantly by one particular subcellular organelle, all the three organelles are involved in the packaging of the maturing CM [also called preCM by Redgrave (94), referring to the intracellular CM prior to release]. For instance, the addition of carbohydrates to newly synthesized protein occurs at both the endoplasmic reticulum and the Golgi apparatus. Using ultrastructural immunocytochemical techniques, Christensen et al. (95) have shown in fasting jejunal biopsies that apo B label was found predominantly at the rough endoplasmic reticulum (RER) and within Golgi cisternae of the absorptive cells at the villus tip. After fat feeding, apo B label was found adjacent to both VLDL and CM within apical smooth endoplasmic reticulum (SER). The apo B label at the rough endoplasmic reticulum becomes less dense, and apo B label was found at the RER and SER junction. They interpreted that apo B is synthesized in the RER and transferred to the SER and is then added onto the lipoprotein particles. Both the Golgi vesicles and the cisternae had apo B label. Assuming the finding of Christensen et al. is correct, this is the first time it has been shown that apo B is present not only on the surface of CM and VLDL, but also at the Golgi vesicle membrane. Whether the apo B on the Golgi vesicle membrane has any physiological significance remains to be demonstrated. In the Golgi apparatus, the preCM undergo considerable modification. Terminal glycosylation occurs at the Golgi apparatus (96). The phospholipid composition of the preCM in the Golgi apparatus also undergoes marked changes, e.g., the en- richment of phosphatidylcholine (94). Nonetheless, we do not know as yet how important these modifications are with regard to the maturation and the subsequent release of the preCM. In the liver, the addition of tunicamycin in the chicken he- patocyte culture medium prevented the glycosylation of both apo B and VLDL apo A-II, but VLDL morphology and secretion were not impaired (97,98). The preCM are then transported in Golgi-derived vesicles (Fig. 1). Through a process of re- verse exocytosis, the preCM are externalized into intercellular space (Fig. 2) (99). The preCM are secreted as a group of particles.

FACTORS REGULATING THE TRANSPORT OF INTESTINAL LIPOPROTEINS

Since protein forms the surface coat of preCM, it is possible that the supply of apolipoprotein is rate limiting for the transport of intestinal CM. As yet the only protein whose biosynthesis seems to be obligatory for the normal transport of CM is apo B. Patients lacking the ability to synthesize apo B failed to transport CM, and the enterocytes in their small intestine are saturated with large lipid droplets (100-102). Steatorrhea and abdominal pain after a fatty meal are common in abetalipoproteinemia patients. Although the experiments with protein synthesis in- hibitors have demonstrated that lipid transport was impaired in animals treated with these agents (103), these studies are complicated by the general effect of these agents on protein synthesis. Under normal physiological conditions there is probably an abundant supply of apo B, and thus it is unlikely that the supply of LIPID ABSORPTION AND METABOLISM IN HUMANS

FIG. 1. After fat ingestion, the cell is filled with numerous fat droplets located within vesicu- lated channels of the smooth endoplasmic reticulum (long arrows). A Golgi zone (G) contains many vesicles filled with nascent chylomicrons measuring 600-3500 A. (x 11,420). (From ref. 99.) apo B is rate-limiting for intestinal CM transport. O'Doherty et al. (104) proposed that luminal PC is important for the intestinal CM formation and transport. This hypothesis has later been confirmed and extended by other studies (105-107). A luminal supply of PC, whether biliary or dietary, is important in sustaining a normal lymphatic fat transport of a large amount of absorbed lipid. Bennett-Clark (108) noted that intraduodenal infusion of PC enhanced both TG and cholesterol output even in bile duct-intact rats. This raises the question whether luminal supply of PC from bile is optimal or rate-limiting. The studies of Strauss and Jacob (109) have demonstrated that Ca2+ is required for the normal transport of CM by the small intestine. The process was impaired UPID ABSORPTION AND METABOLISM IN HUMANS

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<# 1 ' • '9 • ^^ l" r 3^ C4 • iFA* lv 11•i I J IB IB FIG. 2. This electron micrograph shows clearly the release of nascent chylomicrons by the intestinal epithelial cells through exocytosis. The nascent chylomicrons in the secretory vesicles are similar in size and morphology to the chylomicrons present in the intercellular space. (x 24,480). (From ref. 99.)

by replacing H2O with D2O, by lowering temperature below 35°C or by adding lanthanum ions. The precise role of Ca2+ in the intestinal CM transport still remains to be elucidated. Microtubules are probably involved in the movement of preCM-containing Golgi derived vesicles from the Golgi apparatus to the basolat- eral plasma membrane. This notion is supported by the inhibition of intestinal lipid transport by colchicine (110-112). Much work is still needed to elucidate the vari- ous factors regulating the intestinal formation and transport of lipoproteins. Whether hormones, in particular gastrointestinal hormones, play a role in this process still remains to be demonstrated. The ideal system for studying this question is either the isolated enterocytes system or the organ culture system.

LYMPHATIC TRANSPORT OF INTESTINAL CHYLOMICRONS

After the intestinal CM are released into the intercellular space, they still need to penetrate the apparently intact basement membrane, which forms a continuous LIP1D ABSORPTION AND METABOLISM IN HUMANS 9

boundary along the basal portions of the absorptive cells. The morphological study of Sabesin (113) proposed that CM entered the amorphous material of the basement membrane and thereby gained access to the lamina propria. The transport of CM within the lamina propria presumably occurred both by diffusion and also by moving together with the convective fluid movement as a result of water absorption. From the lamina propria the CM entered the central lacteal via gaps in the junctions between adjacent endothelial lining cells of the lymphatics (113). The lymphogogic effect of fat feeding is well established (114,115). However, the physiological importance of this effect is unknown. Recently Tso et al. (116) have presented information showing that the transport of CM from the enterocytes to the central lacteal is greatly influenced by lymph flow. Thus, the lymphogogic effect of fat feeding greatly enhances the transport of CM from the intercellular space to the central lacteal.

METABOLISM OF CHYLOMICRONS IN THE CIRCULATION

As this topic will be discussed at length by A. Bensadoun and B. P. Daggy (this volume), the following is only a brief account of the process involved. Figure 3 shows the apolipoprotein composition of rat mesenteric lymph CM and human chylous urine CM. The major apoproteins present in human CM are apo B, apo A-I, apo A-IV, apo A-II, and apo C. The apo B of human CM is the B-48 as described by Kane et al. (118). It has an apparent molecular weight of approximately 210,000 as determined by sodium dodecylsulfate polyacrylamide gels (SDS-PAGE) (119). This was recently confirmed by the study of Elovson et al. (120) using both sedimentation and diffusion methods.

FIG. 3. Sodium dodecyl sulfate polyacrylamide gels (5.6%) of apolipoproteins of rat (left) mesenteric lymph chylomicrons and human (right) chylous urine chylomicrons. ARP, apo E. An ink marker is present at the bottom of each gel. (From ref. 117.) 10 UP1D ABSORPTION AND METABOLISM IN HUMANS

The CM probably undergo considerable changes when they come into contact with the lipoproteins and apoproteins in the plasma filtrate. For instance, the CM in thoracic duct lymph has a richer apo E and apo C content with the loss of apo A-I and apo A-IV, when compared to the CM harvested from intestinal lymph. These exchanges of apoproteins are completed in the circulation. Through the activation of lipoprotein lipase by apo C-II, the TG is hydrolyzed to glycerol and FA. Lipoprotein lipase is located at the surface of the capillary endothelial cells of adipose tissue, cardiac and skeletal muscle, and other sites. These are then rapidly taken up by the cells. Thus, the CM particles become smaller as the TG is de- pleted. The CM are then converted to CM remnants (121). The remnant particles are rapidly removed by the liver through the apo E receptor (also called the apo E remnant receptor) (122-124).

FUTURE STUDIES

With the ready access of techniques in molecular biology, we hopefully will have a better understanding of the packaging and secretion of intestinal lipoproteins in the human. Through this knowledge we may begin a new era in the control of hyperlipidemia through affecting the intracellular packaging of lipoproteins. Most of the hypolipidemic drugs thus far are agents that work in the intestinal lumen, and some of them are associated with unpleasant side effects (37). Through both dietary and therapeutic means, the rate and the kind (CM versus VLDL) of TG-rich lipoproteins secreted by the small intestine may be regulated in these patients.

ACKNOWLEDGMENTS

This chapter was supported in part by grants from the National Institutes of Health (AM32288, HL30553, K041AM01575, and AM31025). We are extremely grateful to Ms. Becky Potter for help in the preparation of this manuscript.

REFERENCES

1. Davenport HW. Physiology of the digestive tract. 3rd ed. Chicago: Yearbook Medical Publish- ers, 1971. 2. Granger DN, Barrowman JA, Kvietys PR. Clinical gastrointestinal physiology. Philadelphia: WB Saunders Company, 1985:141-207. 3. Hamosh M. The role of lingual lipase in neonatal fat digestion (1979) In: Elliot K, Whelan J, eds. Development of mammalian absorptive process, Ciba Foundation Symposium 70 (new se- ries). Amsterdam: Excerpta Medica, 1979:69-98. 4. Borgstrom B. Phospholipid absorption. In: Rommel K, Goebell H, Bohmer R, eds. Lipid absorption, biochemical and clinical aspects. London: MTP Press Ltd, 1976:65—72. 5. Taylor CB, Gould RG. A review of human cholesterol metabolism. Arch Pathol 1967;84:3-14. 6. Gouid RG, Jones RJ, LeRoy GV, Wissler RW, Taylor CB. Absorbability of p-sitosterol in humans. Metabolism 1969;18:652-62. LIPID ABSORPTION AND METABOLISM IN HUMANS 11

7. Connor WE. In: Hoffman F, ed. Lipid and heart disease. Amsterdam: Excerpta Medica, 1968:44-55. 8. Barrowman JA. In: Csaky TZ, ed. Intestinal absorption of the fat soluble vitamins: physiology and pharmacology. Handbook of experimental pharmacology. Volume 7011. Berlin: Springer Verlag, 1984:647-89. 9. Schonheyder F, Volquartz K. Gastric lipase in man. Acta Physiol Scand 1946; 11:349-60. 10. Senior JR. Intestinal absorption of fats. J Lipid Res 1964;5:495-521. 11. Carey MC, Small DM, Bliss CM. Lipid digestion and absorption. Annu Rev Physiol 1983;45:65I-77. 12. Brockerhoff H. Substrate specificity of pancreatic lipase. Biochim Biophys Acta 1968;159:296- 303. 13. Mattson FH, Beck LW. The specificity of pancreatic lipase or the primary hydroxyl groups of glycerides. J Biol Chem 1956;219:740. 14. Desnuelle P. Pancreatic lipase. Adv Enzymol 1961;23:129-61. 15. Simmonds WJ. The role of micellar solubilization in lipid absorption. Aust J Exp Biol Med Sci 1972;50:403-21. 16. Borgstrom B. On the mechanism of the hydrolysis of glycerides by pancreatic lipase. Acta Chem Scand 1953;7:557-60. 17. Mattson FH, Volpenhein RA. The digestion and absorption of triglycerides. J Biol Chem 1964;239:2772-7. 18. Mattson FH, Volpenhein RA. Hydrolysis of primary and secondary esters of glycerol by pancreatic juice. J Lipid Res 1968;9:79-84. 19. Borgstrom B. Influence of bile salt, pH and time on the action of pancreatic lipase; physiological implications. J Lipid Res 1964;5:522-31. 20. Hofmann AF, Borgstrom B. Hydrolysis of long-chain monoglycerides in micellar solution by pancreatic lipase. Biochim Biophys Acta 1963;70:317-31. 21. Benzonana G, Desnuelle P. Action of some effectors on the hydrolysis of long-chain triglycerides by pancreatic lipase. Biochim Biophys Acta 1968; 164:47-58. 22. Borgstrom B, Erlanson C. Pancreatic lipase and colipase. Interactions and effects of bile salts and other detergents. Eur J Biochem 1973;37:60-8. 23. Morgan RGH, Barrowman J, Borgstrom B. The effect of sodium taurodeoxycholate and pH on the gel filtration behaviour of rat pancreatic protein and lipases. Biochim Biophys Acta 1969;175:65-75. 24. Maylie MF, Charles M, Gache C, Desnuelle P. Isolation and partial identification of a pancreatic colipase. Biochim Biophys Acta 1971;229:286-9. 25. Borgstrom B, Erlanson-Albertson C. Hydrolysis of milk fat globules by pancreatic lipase. Role of colipase, phospholipase A2, and bile salts. J Clin Invest 1982;70:30-2. 26. Borgstrom B, Erlanson-Albertson C, Wieloch T. Pancreatic colipase—chemistry and physiology. J Lipid Res 1979;20:805-16. 27. Wieloch T, Borgstrom B, Pieroni G, Pattus F, Verger R. Porcine pancreatic procolipase and its trypsin-activated form: lipid binding and lipase activation on monomolecular films. FEBS Lett 1981;128:217-20. 28. Arnesjo B, Barrowman J, Borgstrom B. The zymogen of phospholipase A2 in rat pancreatic juice. Acta Chem Scand 1967;21.2887-900. 29. Arnesjo B, Nilsson A, Barrowman J, Borgstrom B. Intestinal digestion and absorption of cholesterol and lecithin in the human. Scand J Gastroenterol 1969;4:653-65. 30. Scow RO, Stein Y, Stein O. Incorporation of dietary lecithin and lysolecithin into lymph chylomicrons in the rat. J Biol Chem 1967;242:4919-24. 31. Parthasarathy S, Subbaiah PV, Ganguly J. The mechanism of intestinal absorption of phosphatidylcholine in rats. Biochem J 1974;140:503-8. 32. Boucrot P, Clement J. Participation des acides gras du sang et de la bile a la formation des lipides endogenes de la lymphe chez le rat qui ingere un repas contenant des graisses. Biochim Biophys Acta 1969;187:59-72. 33. Boucrot P, Clement JR. Resistance to the effect of phospholipase A2 of the biliary phospholipids during incubation of the bile. Lipids 1971;6:652—6. 34. Boucrot P. Is there an entero-hepatic circulation of the bile phospholipids? Lipids 1972;7: 282-8. 35. Swell L, Field H Jr, Treadwell CR. Absorption of cholesterol 4-l4C oleate. Proc Soc Exp Biol Med 1960;103:263-6. 12 LIPID ABSORPTION AND METABOLISM IN HUMANS

36. Hyun J, Kothari H, Herm E, Mortenson J, Treadwell CR, Vahouny GV. Purification and properties of pancreatic juice cholesterol esterase. J Biol Chem 1969;244:1937—45. 37. Hofmann AF. Fat digestion: the interaction of lipid digestion products with micellar bile acid solutions. In: Rommell K, Goebell H, Bohmer R, eds. Lipid absorption, biochemical and clinical aspects. Lancaster: MTP Press Ltd, 1976;3-18. 38. Patton JS, Carey MC. Watching fat digestion. Science 1979;204:145-8. 39. Stafford RJ, Donovan JM, Benedek GB, Carey MC. Physical-chemical characteristics of aqueous duodenal content after a fatty meal. Gastroenterology 1980;80:1291. 40. Stafford RJ, Carey MC. Physical-chemical nature of the aqueous lipids in intestinal content after a fatty meal: Revision of the Hofmann—Borgstrom hypothesis. Clin Res 1981;28:511A. 41. Mansbach CM, Newton D, Stevens RD. Fat digestion in patients with bile acid malabsorption but minimal steatorrhea. Dig Dis Sci 198O;25:353-62. 42. Simmonds WJ. Uptake of fatty acid and monoglyceride. In: Rommel K, Goebell H, Bohmer R, eds. Lipid absorption, biochemical and clinical aspects. Lancaster: MTP Press Ltd, 1976;51-64. 43. Thomson ABR, Dietschy JM. Intestinal lipid absorption: Major extracellular and intracellular events. In: Johnson LR, ed. Physiology of the gastrointestinal tract. New York: Raven Press, 1981:1147-220. 44. Wilson FA, Sallee VL, Dietschy JM. Unstirred water layers in intestine: Rate determinant of fatty acid from micellar solutions. Science 1971; 174:1031—3. 45. Dietschy JM, Sallee VL, Wilson FA. Unstirred water layers and absorption across the intestinal mucosa. Gastroenterology 1971;61:932—4. 46. Dietschy J. Mechanisms of bile acid and fatty acid absorption across the unstirred water layer and brush border of the intestine. Helv Med Ada 1973;17:89-102. 47. Hoffman NE, Yeoh VJ. The relationship between concentration and uptake by rat small intestine, in vitro, for two micellar solutes. Biochim Biophys Acta 1971;233:49-52. 48. Mclntyre N. Cholesterol absorption. In: Rommel K, Goebell H, Bohmer R, eds. Lipid absorption: biochemical and clinical aspects. Lancaster: MTP Press Ltd, 1976:73-84. 49. Sylven C. Influence of blood supply on lipid uptake from micellar solutions by the rat small intestine. Biochim Biophys Acta 1970;203:365-75. 50. Brindley DN, Hubscher G. The intracellular distribution of the enzymes catalyzing the biosynthesis of glycerides in the intestinal mucosa. Biochim Biophys Acta 1965;lO6:495-509. 51. Schiller CM, David JSK, Johnston JM. The subcellular distribution of triglyceride synthetase in the intestinal mucosa. Biochim Biophys Acta 1970;210:489-99. 52. Ockner RK, Manning JA, Poppenhauser RB, Ho WKL. A binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium and other tissues. Science 1972;177:56-8. 53. Ockner RK, Manning JA. Fatty acid-binding protein in small intestine. Identification, isolation and evidence for its role in cellular fatty acid transport. J Clin Invest 1974;54:326-38. 54. Rao GA, Johnston JM. Purification and properties of triglyceride synthetase from the intestinal mucosa. Biochim Biophys Acta 1966;125:465—73. 55. Raghaven SS, Ganguly J. Studies on the positional integrity of glyceride fatty acids during digestion and absorption. Biochem J 1969;! 13:81—7. 56. Kayden HJ, Senior JR, Mattson FH. The monoglyceride pathway of fat absorption in man. J Clin Invest 1967;46:1695-703. 57. Johnston JM. Esterification reactions in the intestinal mucosa and lipid absorption. In: Dietschy JM, Gotto AM, Outko JA, eds. Disturbances in lipid and lipoprotein metabolism. Bethesda: American Physiological Society, 1978:57—68. 58. Gordon SG, Kern F Jr. The effect of taurocholate on jejunal glyceride synthesis. Gastroenterol- ogy 1970;58:953A. 59. Polheim D, David JSK, Schultz FM, Wylie MB, Johnston JM. Regulation of triglyceride biosynthesis in adipose and intestinal tissue. J Lipid Res 1973;14:415—21. 60. Johnston JM, Paltouf F, Schiller CM, Schultz LD. The utilization of the a-glycerophosphate and monoglyceride pathways for phosphatidylcholine biosynthesis in the intestine. Biochim Bio- phys Acta 1970;218:124-33. 61. Mansbach CM, Parthasarathy S. A re-examination of the fate of glyceride-glycerol in neutral lipid absorption and transport. J Lipid Res 1982;23:1009-19. 62. Lands WEM. Metabolism of glycerolipids: the enzymatic acylation of lysolecithin. J Biol Chem 1960;235:2233-7. 63. Nilsson A. Intestinal absorption of lecithin and lysolecithin by lymph fistula rats. Biochim Bio- phys Acta 1968;152:379-90. UPID ABSORPTION AND METABOUSM IN HUMANS 13

64. Ottolenghi A. Estimation and subcellular distribution of lecithinase activity in rat intestinal mucosa. J Lipid Res 1964;5:532-7. 65. Subbaiah PV, Ganguly J. Studies on the phospholipases of rat intestinal mucosa. Biochem J 1970;l 18:233-9. 66. Simmonds WJ. Fat absorption and chylomicron formation. In: Nelson GJ, ed. Blood lipids and lipoproteins: quantitation, composition and metabolism. New York: Wiley (Interscience), 1972:705-43. 67. Quintao EC, Drewiacki A, Steckhaln K, De Faria EC, Sipahi AM. Origin of cholesterol transport in intestinal lymph: studies in patients with filarial chyluria. J Lipid Res 1979;20:941-51. 68. Stange EF, Suckling KE, Dietschy JM. Synthesis and coenzyme A-dependent esterification of cholesterol in rat intestinal epithelium. J Biol Chem 1983;258:12868-75. 69. Harold G, Schneider A, Difschuneit H, Stange EF. Cholesterol synthesis and esterification in cultured intestinal mucosa. Biochim Biophys Ada 1984;796:27-33. 70. Borja CR, Vahouny GV, Treadwell CR. Role of bile and pancreatic juice in cholesterol absorption and esterification. Am J Physiol 1964;206:223-8. 71. Gallo LL, Newbill T, Hyun J, Vahouny GV, Treadwell CR. Role of pancreatic cholesterol esterase in the uptake and esterification of cholesterol by isolated intestinal cells. Proc Soc Exp Biol Med 1977;156:277-81. 72. Gallo LL, Chiang Y, Vahouny GV, Treadwell CR. Localization and origin of rat intestinal cholesterol esterase determined by immunocytochemistry. J Lipid Res 1980;21:537-45. 73. Watt SM, Simmonds WJ. The effect of pancreatic diversion on lymphatic absorption and esterification of cholesterol. J Lipid Res 1981;22:157-65. 74. Heider JG, Pickens CE, Kelly LA. Role of acyl-CoAxholesterol acyltransferase in cholesterol absorption and its inhibition by 57-118 in the rabbit. J Lipid Res 1983;24:1127-34. 75. Norum KR, Lilljequist AC, Helgerud P, Normann ER, Mo A, Selbekk B. Esterification of cholesterol in human small intestine: the importance of acyl-CoA:cholesterol acyltransferase. Eur J Clin Invest 1979;9:55-62. 76. Norum KR, Helgerud P, Petersen LB, Groot PHE, DeJonge HR. Influence of diets on acyl- CoA:cholesterol acyltransferase and on acyl-CoA:retinol acyltransferase in villous and crypt cells from rat small intestinal mucosa and in the liver. Biochim Biophys Acta 1983;751:153—61. 77. Grundy SM, Ahrens EH Jr, Salen G. Dietary P-sitosterol as an internal standard to correct for cholesterol loss in sterol balance studies. J Lipid Res 1968;9:374-89. 78. Borgstrom B. Quantification of cholesterol absorption in man by faecal analysis after the feeding of a single-isotope labelled meal. J Lipid Res 1969;10:331-7. 79. Salen G, Ahrens EH Jr, Grundy SM. Metabolism of f$-sitosterol in man. J Clin Invest 1970;49:952-67. 80. Bhattacharyya AK, Connor WE. f5-sitosterolemia and xanthomatosis: a newly described lipid storage disease in two sisters. J Clin Invest 1974;53:1033-43. 81. Connor WE, Lin DS. The intestinal absorption of dietary cholesterol by hypercholesterolaemic and normal humans. J Lab Clin Med 1970;76:870A. 82. Quintao E, Grundy SM, Ahrens EH Jr. An evaluation of four methods of measuring cholesterol absorption by the intestine in man. J Lipid Res 1971;12:221-32. 83. Kudchodkar BH, Sodhi HS, Horlick L. Absorption of dietary cholesterol in man. Metabolism 1973;22:155-63. 84. Borgstrom B, Radner S, Wemer B. Lymphatic transport of cholesterol in the human being. Ef- fect of dietary cholesterol Scand J Lab Clin Invest 197O;26:227-35. 85. Beveridge JMR, Connell WF, Haust HL, Mayer GA. Dietary cholesterol and plasma cholesterol levels in man. Can J Biochem 1959;37:575-82. 86. Connor WE, Hodges RE, Bleiler RE. The serum lipids in men receiving high cholesterol and cholesterol-free diet. J Clin Invest 1961;40:894-901. 87. Connor WE, Stone DB, Hodges RE. The interrupted effects of dietary cholesterol and fat upon human serum lipid levels. J Clin Invest 1964;43:1691-6. 88. Glickman RM, Green PHR, Lees RS, Tall A. Apolipoprotein A-I synthesis in normal intestinal mucosa and in Tangier's disease. N Engl J Med 1978;299:1424-7. 89. Lackner KJ, Edge SB, Gregg RE, Hoeg JM, Brewer HB Jr. Isoforms of proapolipoproteins A- II and sialic acid-containing isoforms. J Biol Chem 1985;260:703-6. 90. Green PHR, Glickman RM. Human intestinal lipoproteins. Studies in chyluric subjects. J Clin Invest 1979;64:233-42. 14 UPID ABSORPTION AND METABOLISM IN HUMANS

91. Gordon JI, Bisgaier CL, Sims HF, Sachdev DP, Glickman RM, Strauss AW. Biosynthesis of human preapolipoproteins A-IV. J Biol Chem 1984;259:468-74. 92. Glickman RM, Green PHR, Lees RS, Lux SE, Kilgore A. Immunofluorescence studies of apolipoprotein B in intestinal mucosa: absence in abetalipoproteinemia. Gastroenterology 1979; 76:288-92. 93. Kessler J, Narcessian P, Mauldin DP. Biosynthesis of lipoproteins by intestinal epithelium. Site of synthesis and sequence of association of lipid, sugar and protein moieties. Gastroenterology 1972;68:1058A. 94. Redgrave TG. Association of Golgi membrane with lipid droplets (pre-chylomicrons) from within intestinal epithelial cells during absorption of fat. Aust J Exp Biol Med Sci 1971;49:209— 24. 95. Christensen NJ, Rubin CE, Cheung MC, Albers JJ. Ultrastructural immunolocalization of apolipoprotein B within human jejunal absorptive cells. J Lipid Res 1983;24:1229-42. 96. Leblond CP, Bennett G. In: Brinkley BR, Porter KR. ed. International cell biology. New York: Rockefeller University Press, 1977:326-36. 97. Siuta-Mangano P, Howard S, Lennarz WJ, Lane MD. Synthesis, processing, and secretion of apolipoprotein B by the chick liver cell. J Biol Chem 1982;257:4292-300. 98. Janero DR, Siuta-Mangano P, Miller KW, Lane MD. Synthesis, processing and secretion of hepatic very low density lipoproteins. J Cell Biochem 1984;24:131-52. 99. Sabesin SM, Frase S. Electron microscopic studies of the assembly, intracellular transport, and secretion of chylomicrons by rat intestine. J Lipid Res 1977; 18:496-511. 100. Isselbacher KJ, Scheig R, Plotkin GR, Caulfield JR. Congenital |J-lipoprotein deficiency: a he- reditary disorder involving a defect in the absorption and transport of lipids. Medicine 1964;43:347-61. 101. Fredrickson DS, Gotto AM, Levy RI. Familial lipoprotein deficiency. In: Stanbury JB, Wyn- gaarden JB, Frederickson DS, eds. The metabolic basis of inherited diseases. New York: Mc- Graw-Hill, 1972:493-530. 102. Kayden HJ. Abetalipoproteinemia. Annu Rev Med 1972;23:285-96. 103. Sabesin SM, Isselbacher KJ. Protein synthesis inhibition: mechanism for the production of impaired fat absorption. Science 1965; 147:1149-51. 104. O'Doherty PJA, Kakis G, Kuksis A. Role of luminal lecithin in intestinal fat absorption. Lipids 1973;8:249-55. 105. Tso P, Balint JA, Simmonds WJ. Role of biliary lecithin in lymphatic transport of fat. Gastroen- terology 1977;73:1362-7. 106. Tso P, Lam J, Simmonds WJ. The importance of the lysophosphatidylcholine and choline moiety of bile phosphatidylcholine in lymphatic transport of fat. Biochim Biophys Acta 1978;528:364-72. 107. Tso P, Kendrick H, Balint JA, Simmonds WJ. Role of biliary phosphatidylcholine in the absorption and transport of dietary triolein in the rat. Gastroenterology 1981;80:60-5. 108. Bennett-Clark S. Chylomicron composition during duodenal triglyceride and lecithin infusion. Am J Physiol 1978;235:E183-90. 109. Strauss EW, Jacob JS. Some factors affecting the lipid secretory phase of fat absorption by intestine in vitro from golden hamster. J Lipid Res 1981;22:147-56. 110. Glickman RM, Perotto JL, Kirsch K. Intestinal lipoprotein formation: effect of colchicine. Gas- troenterology 1976;70:347-52. 111. Arreaza-Plaza CA, Bosch V, Otayek MA. Lipid transport across the intestinal epithelial cell. Effect of colchicine. Biochim Biophys Acta 1976;431:297-302. 112. Pavelka M, Gangl A. Effects of colchicine on the intestinal transport of endogenous lipid. Gas- troenterology 1983;84:544-55. 113. Sabesin SM. Ultrastructural aspects of the intracellular assembly, transport and exocytosis of chylomicrons by rat intestinal absorptive cells. In: Rommel K, Goebell H, Bohmer R, eds. Lipid absorption, biochemical and clinical aspects. Lancaster: MTP Press Ltd, 1976:113-48. 114. Borgstrom B, Laurell CB. Studies on lymph and lymph-proteins during absorption of fat and saline by rats. Acta Physiol Scand 1953;29:264-80. 115. Simmonds WJ. Some observations on the increase in thoracic duct lymph flow during intestinal absorption of fat in unanesthetized rats. Aust J Exp Biol Med Sci 1955;33:3O5-13. 116. Tso P, Pitts V, Granger DN. Role of lymph flow in intestinal chylomicron transport. Am J Phys- iol 1985;249:G21-8. LIP1D ABSORPTION AND METABOLISM IN HUMANS 15

117. Bisgaier CL, Glickman RL. Intestinal synthesis, secretion, and transport of lipoproteins. Ann Rev Physiol 1983;45:625-36. 118. Kane JP, Hardman DA, Paulus HE. Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons. Proc Natl Acad Sci USA 1980;77:2465-9. 119. Kane JP. Apolipoprotein B: structural and metabolic heterogeneity. Annu Rev Physiol 1983;45:637-50. 120. Elovson J, Jacobs JC, Schumaker VN, Puppione DL. Molecular weight of apoprotein B ob- tained from human low-density lipoproteins (apoprotein B-PI) and from rat very low density lipoprotein (apoproprotein B-PIII). Biochemistry 1985;24:1569-78. 121. Redgrave TG. Formation of cholesteryl ester-rich particulate lipid during metabolism of chylomicrons. J Clin Invest 1970;49:465-71. 122. Quarfordt SH, Hanks J, Jones RS, Shelburne F. The uptake of high density lipoprotein cholesteryl ester in the perfused rat liver. J Biol Chem 1980;255:2934-7. 123. Sherrill BC, Innerarity TL, Mahley RW. Rapid hepatic clearance of the canine lipoproteins containing only the E apoprotein by a high affinity receptor. J Biol Chem 1980;255:1804-7. 124. Mahley RW, Hui DY, Innerarity TL, Weisgraber KH. Two independent lipoprotein receptors on hepatic membranes of the dog, swine, and man. Apo B, E and apo-E receptors. J Clin Invest 1981;68:1197-206.