Enzymatic hydrolysis and micellar solubilization of dietary lipids. BA conjugated bile acid, C cholesterol, CE cholesterol ester, DG diacylglycerol, FFA free fatty acid, LPC lysophosphatidylcholine, MG monoacylglycerol, PC phosphatidylcholine, TG triacylglycerol, V fat-soluble vitamin, VE fat-soluble vitamin ester
of lipids and lipolytic products due to the formation of mixed micelles is a critical step for the absorption. Conjugated bile acids play a key role in this process, but polar lipids, including fatty acids and monoglycerides, are also important to increase the solubility of nonpolar lipids such as cholesterol. In contrast to emulsion, mixed micelle solution is optically clear. The diameter of the mixed micelles is 4–5 nm, which means that they have an approximately 100-fold reduced size and 10,000-fold increased surface area relative to the fine emulsion particle. It is also estimated that one fine emulsion particle can form approximately 1 × 106 micelles . The unstirred water layer overlying the microvillus border of the epithelial cells is an intestinal diffusion barrier (Fig. 3.2). The mixed micelles effectively pass into the intermicrovillous spaces and are able to reach to the epithelial cells . However, it seems to be an oversimplification to divide the intestinal phase of lipids into an emulsion and mixed micelles. Under an adequate concentration of bile acids, the lipids are incorporated into mixed micelles. When the bile acid concentration is relatively low but still exceeds the critical micellar concentration, large mixed disclike micelles are formed at approximately 40 nm in diameter. Furthermore, when the relative bile acid concentration is much lower, this results in the formation of vesicles (liposomes) with a diameter of approximately 80–120 nm . Because patients with low upper intestinal bile acid concentration show reasonably good absorption of lipids [25, 26], vesicles may play an important role in the uptake of free fatty acids and monoglycerides by enterocytes . However, the relative roles of the mixed micelles and the vesicles have not been clarified .
Micellar solubilization and absorption of lipids in the upper small intestine. ABCG5/ABCG8 ATP-binding cassette G5 and G8, BA conjugated bile acid, C cholesterol, CD36 cluster determinant 36, FFA free fatty acid, LPC lysophosphatidylcholine, MG monoacylglycerol, NPC1L1 Niemann-Pick C1-like 1, PC phosphatidylcholine, SR-BI scavenger receptor class B type I, V fat-soluble vitamin
Most of the triacylglycerols found in food have long-chain fatty acids with 16–18 carbon atoms. However, a small but variable proportion of triacylglycerols contain fatty acids with only 6–10 carbon atoms and are called medium-chain triacylglycerols . Because medium-chain triacylglycerols are less hydrophobic than long-chain triacylglycerols, the processes of digestion and absorption of these two types of triacylglycerols are somewhat different.
184.108.40.206 Long-Chain Triacylglycerols
The digestion of dietary triacylglycerols begins in the stomach. In addition to emulsification, gastric lipase hydrolyzes a significant portion of dietary triacylglycerols. This enzyme hydrolyzes medium-chain triacylglycerols better than long-chain triacylglycerols  and preferentially acts on the sn-3 position of the triacylglycerols  to release diacylglycerols and free fatty acids [19, 20, 30]. The relative contributions of gastric lipase and pancreatic triacylglycerol lipase to the hydrolysis of dietary triacylglycerols were reported to be approximately 1:3, and approximately 40% of the hydrolysis by gastric lipase occurred in the duodenum .
In the duodenum and proximal jejunum, the rest of triacylglycerols and diacylglycerols are hydrolyzed by pancreatic triacylglycerol lipase. This enzyme works at the oil-water interface of the emulsion. Conjugated bile acids adsorb onto fat droplets and remove proteins, emulsifiers, and lipolysis products from the lipid surface . However, only triacylglycerol lipase is not removed from the oil-water interface and instigates lipolysis because colipase binds a bile-acid-covered oil-water interface and provides a high-affinity anchor site for triacylglycerol lipase [5, 21, 32]. Pancreatic triacylglycerol lipase preferentially cleaves the ester bond in the sn-1 and sn-3 positions of the triacylglycerols at equal rates [29, 33] so that 2-monoacylglycerol and free fatty acids are formed. A part of 2-monoacylglycerol is further hydrolyzed into glycerol and a free fatty acid by pancreatic triacylglycerol lipase either directly or after isomerization to 1-monoacylglycerol [33, 34]. Carboxyl ester lipase also hydrolyzes the acyl group at the sn-2 position to release glycerol and free fatty acid . However, 80–90% of dietary glycerides retain their fatty acid in the sn-2 position during the entire digestion and absorption process .
Long-chain triacylglycerols and diacylglycerols are insoluble in aqueous solution regardless of whether bile acids are present . Therefore, most of these acylglycerols reside in emulsified oil phase. In contrast, monoacylglycerols and free fatty acids possess polar groups that make them highly soluble in the presence of conjugated bile acids to form mixed micelles. The mixed micelles effectively pass through the unstirred water layer overlying the microvillus border of the enterocytes, and monoacylglycerols and free fatty acids are finally taken up by the cells .
The mechanisms by which free fatty acids and monoacylglycerols are translocated into the enterocytes have not been elucidated completely. Cluster determinant 36 (CD36) or fatty acid translocase (FAT) is known to be a membrane protein that facilitates cellular uptake of long-chain fatty acids. This protein is also highly expressed on the luminal surface of enterocytes in the proximal small intestine [36, 37]. However, CD36-null mice exhibited normal overall absorption of long-chain fatty acids and impaired chylomicron secretion. These findings suggest that CD36 plays critical roles for the absorption of long-chain fatty acids and the formation of chylomicron in the proximal small intestine, but CD36-independent absorption mechanisms predominate in the distal segments . In comparison to free fatty acids, studies on intestinal uptake of monoacylglycerols are limited. An in vitro study using human intestinal Caco-2 cells showed that long-chain fatty acid and 2-monoacylglycerol were taken up in a saturable and competitive manner. The results suggest that long-chain fatty acids and 2-monoacylglycerol are transported into the enterocyte, at least in part, via a protein-mediated pathway that is shared by both lipids .
220.127.116.11 Medium-Chain Triacylglycerols
Higher concentrations of medium-chain length fatty acids are found in coconut oil (14%) and palm kernel oil (7%), butter (3%), and fresh cream (2%); cow and breast milk fat (1–3%) also contain significant amounts of the fatty acids . However, the ingestion of medium-chain fatty acids is reported to be less than 2% of the total fatty acid intake in the United States .
Gastric lipase and pancreatic triacylglycerol lipase work more efficiently with medium-chain triacylglycerols than long-chain triacylglycerols. As a consequence, medium-chain triacylglycerols are absorbed mainly as free fatty acids and glycerol and only rarely as mono- or diacylglycerols . Because of their smaller molecular size, medium-chain fatty acids and glycerol have greater solubility in water, and micellization with bile acids is unnecessary. In contrast to long-chain fatty acids that are resynthesized to triacylglycerol in the enterocytes and follow the lymphatic system as chylomicrons, medium-chain fatty acids are bound with albumin and follow the portal venous system .
Dietary phospholipids are not hydrolyzed by gastric lipase but aid the emulsification of dietary fat. Therefore, they are forwarded to the duodenum as a component of emulsified oil droplets. In contrast, biliary phospholipids (essentially phosphatidylcholine) are supplied in mixed micelles along with cholesterol and conjugated bile acids. In the upper small intestine, dietary phospholipids are redistributed much in favor of the micellar phase .
The digestion of phospholipids is carried out mainly by pancreatic phospholipase A2, but carboxyl ester lipase may contribute to the hydrolysis of phosphatidylcholine and lysophosphatidylcholine to some extent . In fact, mice deficient in phospholipase A2 show no abnormality in dietary phospholipid absorption . Phospholipase A2 undergoes a substantial increase in the catalytic activity on binding to the surface of phospholipid membranes or micelles  and the presence of bile acids . However, the enzyme shows a low activity on biliary phosphatidylcholine because the high bile acid/phosphatidylcholine molar ratio in native bile presents unfavorable conditions for hydrolysis . Phospholipase A2 preferentially cleaves the ester bond in the sn-2 position of the phospholipids to yield lysophosphatidylcholine and free fatty acid .
Deacylation of lysophosphatidylcholine in the gut lumen is believed to be quite limited, and lysophosphatidylcholine and free fatty acids are taken up by enterocytes and resynthesized to phospholipids or triacylglycerols, which follow the lymphatic system as chylomicrons. The remaining absorbed lysophosphatidylcholine is hydrolyzed to form glycero-3-phosphorylcholine by phospholipase A2/lysophospholipase (phospholipase B) [48–50], which is readily transported via the portal blood for use in the liver . Although specific intestinal transporters for phosphatidylcholine and lysophosphatidylcholine have not been identified, lysophosphatidylcholine uptake by enterocytes is much greater than phosphatidylcholine absorption [52, 53].
3.3.3 Cholesterol and Plant Sterols
Most dietary cholesterol is present in the free form, but 10–15% exists as cholesterol ester . Gastric lipase does not hydrolyze cholesterol ester; rather, the hydrolysis is performed by pancreatic carboxyl ester lipase (cholesterol esterase). In this process, bile acids strongly stimulate the lipase activity [54, 55]. Chemical modification studies suggest that positive-charged arginine residues in carboxyl ester lipase are important for its interaction with bile acids [56–59, 22]. In contrast, biliary cholesterol is exclusively free form and is secreted as mixed micelles with phosphatidylcholine and conjugated bile acids. In the proximal small intestine, dietary cholesterol is initially emulsified with triglycerides in oil droplets, but free cholesterol originated from the diet is finally incorporated into mixed micelles or vesicles with biliary cholesterol .
In humans, cholesterol absorption is not complete, and the percent of absorption varies from 15% to 75% . Relative to monoacylglycerols, free fatty acids, and lysophosphatidylcholine, the aqueous solubility of cholesterol is extremely low. Therefore, the formation of mixed micelles and vesicles is critically important for the transport of cholesterol through the unstirred water layer overlying the microvillus border of enterocytes. Therefore, intestinal cholesterol absorption is markedly affected by coexisting bile acids, phospholipids, free fatty acids, and plant sterols.
It has been reported that trihydroxy bile acids more effectively promote cholesterol absorption than dihydroxy bile acids [62–64], and the size of the cholic acid pool significantly correlates with cholesterol absorption in patients with liver cirrhosis . On the other hand, the intestinal uptake of cholesterol was linearly dependent on micellar cholesterol concentration and was not dependent on the bile acid concentration . However, taurochenodeoxycholic acid is a better micellar solubilizer of cholesterol than taurocholic acid, although the latter is a better promoter of cholesterol absorption [66–68]. In addition, when cholesterol was completely solubilized in micelles with a nontoxic nonionic detergent, Pluronic F68, cholesterol was not taken up by enterocytes . These results suggest that not only the solubilization capacity but also the interaction between micelle and acceptor (transporter) serves as determinants of the absorption efficiency of cholesterol.
There are at least four transporters that are key players in the control of cholesterol absorption from the intestine. Niemann-Pick C1-like 1 (NPC1L1) is a major cholesterol uptake transporter , while scavenger receptor class B type I (SR-BI) also plays a role in cholesterol uptake to a lesser extent [70, 71]. On the other hand, ATP-binding cassette (ABC) proteins ABCG5 and ABCG8 are cholesterol efflux transporters . Although little is known about the direct effects of bile acids on intestinal NPC1L1 and SR-BI activities, ABCG5-/ABCG8-specific cholesterol efflux is stimulated by bile acids in cell models [73, 74]. ABCG5/ABCG8 transfers cholesterol in an ATP-dependent manner, and the hydrolysis of ATP is stimulated by bile acids . It has been suggested that bile acids may promote an active conformation of ABCG5/ABCG8 either by global stabilization of the transporter or by binding to a specific site on ABCG5/ABCG8. Furthermore, CD36 may also play a role in cholesterol uptake. Overexpression of CD36 enhanced cholesterol uptake from micellar substrates in COS-7 cells . Conversely, CD36-null mice showed significant reduced cholesterol transport from the intestinal lumen to the lymphatic system . However, its absence was not sufficient to cause an overall reduction in intestinal cholesterol uptake.
Although phospholipids are essential molecules for the effective solubilization of cholesterol in the bile and intestine, excess phospholipids cause the suppression of cholesterol absorption. There are at least three possible mechanisms . First, excess phospholipids may interfere with efficient hydrolysis of micellar phospholipids, which is a prerequisite for efficient mucosal uptake of cholesterol. Second, surplus phospholipids may alter the physicochemical properties of mixed micelles resulting in reduced absorption of cholesterol. Third, phospholipids may act on the membrane characteristics of enterocytes or have a direct effect on cellular cholesterol transporters. Free fatty acids may also affect intestinal cholesterol absorption. Mixed micelles containing medium-chain fatty acids have a reduced solubilizing capacity for cholesterol relative to those containing long-chain fatty acids .
Food of a plant origin includes plant sterols that are structurally related to cholesterol but differ from cholesterol only in their unsaturation level and/or side-chain configuration . Typical Western diets contain approximately 300 mg of plant sterols per day [79, 80], but the absorption percentage of plant sterols is less than 2% in humans , which is considerably lower than that of cholesterol (15–75%). Plant sterols transported into enterocytes with cholesterol via NPC1L1 are pumped back to the lumen via ABCG5/ABCG8, whereas a significant proportion of the internalized cholesterol is esterified and incorporated into chylomicrons [82, 83]. Plant sterols are known to inhibit cholesterol absorption, but the mechanisms are not fully understood. Because the digestion process of plant sterols and cholesterol is virtually the same, it has been suggested that plant sterols compete with intestinal cholesterol for incorporation into mixed micelles [84, 85], but other possible mechanisms have also been proposed .
3.3.4 Fat-Soluble Vitamins
Because vitamins A, D, E, and K are fat soluble, micelle formation is required for intestinal absorption. Vitamins A, D, and E have hydroxyl groups that can be esterified with fatty acid, and pancreatic carboxyl ester lipase catalyzes the hydrolysis under the presence of bile acids . While most dietary vitamin E (tocopherol and tocotrienol) is in the free form, vitamin A (retinol) is often esterified and must be hydrolyzed to retinol and fatty acid before absorption [22, 87]. Except for carboxyl ester lipase, pancreatic triacylglycerol lipase and intestinal phospholipase B also contribute to the hydrolysis of retinyl esters . Free retinol is then incorporated with other lipids into the mixed micelles, passes through the unstirred water layer, and is taken up by enterocytes . Although the intestinal retinol-specific transporter has not been clarified, stimulated by retinoic acid 6 (STRA6)  and retinol-binding protein 4-receptor 2 (RBPR2)  are candidate proteins.
Micelle formation is also required for the absorption of vitamins E, D, and K [12, 90]. It has been reported that intestinal cholesterol transporters, NPC1L1 and SR-BI, play a role in the uptake of micellar vitamin E [91, 92]. However, recent report suggests that additional intestinal transporters are also involved in the uptake of vitamin E . In addition, there are reports that both SR-BI and CD36 contribute to the intestinal absorption of vitamins D  and K .
3.4 Protein Digestion
In addition to promoting lipid digestion and absorption, conjugated bile acids also bind to dietary proteins in the small intestine. The binding of bile acids denatures the protein and dramatically enhances the proteolysis by pancreatic proteases . The effect was most pronounced in the presence of dihydroxy bile acids and was observed at concentrations below the critical micellar concentration.
3.5 Absorption of Polyvalent Metals
Polyvalent metals such as calcium (Ca2+) and iron (Fe2+) are poorly soluble at the intestinal pH. However, premicellar concentrations of taurocholic acid solubilize calcium  and iron [98, 99] in the proximal small intestine and promote their absorption. The mechanism of solubilization is explained by high-affinity binding of these polyvalent cations to the interposition between terminal carboxyl and 7- or 12-hydroxyl groups of the steroid ring of taurocholic acid. Taurodehydrocholic acid, lacking ring hydroxyl groups, did not bind either cation with a high affinity and did not promote their absorption .
In the duodenum and upper jejunum, the conjugated bile acids facilitate lipolysis by pancreatic lipases and formation of mixed micelles with phospholipids, lipolytic products of triacylglycerols (2-monoacylglycerols and free fatty acids), cholesterol, and fat-soluble vitamins. The mixed micelles can effectively approach the enterocytes, and each nutrient is finally taken up by the cells primarily by protein-mediated processes. Conjugated bile acids also promote digestion of proteins and absorption of polyvalent metals, such as calcium and iron. In addition to these direct effects on digestion and absorption, duodenal conjugated bile acids are known to inhibit the release of cholecystokinin  and motilin , which modulate contractions of the gallbladder and indirectly control digestion and absorption. Conjugated bile acids maintain water solubility at an acidic pH in the upper small intestine and are not absorbed together with the solubilized lipids, which allows for efficient active absorption of bile acids from the terminal ileum after the completion of their roles.
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