IDL are generated in blood by TG depletion of the VLDL particles. They can be taken up by the liver or by hydrolysis of TG via hepatic lipase (HL). They can be transformed into LDL. LDL are formed in blood by delipidation of VLDL and HDL by LPL and HL. They account for >70% of the cholestreol present in human blood. Apo B-100 is the only apolipoprotein on the surface of LDL. A structure related to LDL is known as lipoprotein (a), Lp(a). Lp(a) has additional lipoprotein ,apo(a), bound to its apoB, which is thought to be an atherogenic and thrombogenic particle.
HDL are secreted by the liver and intestine as lipid -poor apoA-I that become immediately lipidated by the action of the ATP-binding cassette transporter A1. Peripheral cells are unable to degrade cholestrol . mature HDL particles are formed by incorporation of such cholestrol after esterification by lecithin -cholestrol acyl transferase(LCAT). Cholesterol ester transfer protein(CETP) in plasma is useful for the exchange of CE for triglycerides between HDL and apoB- containing lipoproteins . HDL promote expulsion of cholesterol from peripheral cells(RCT)and indirectly, from the body, thus protecting against cardiovascular diseases.
1.7 Intestinal cholesterol absorption
Cholestrol enters the lumen of the small intestine by four routes,
The bile
The diet
The intestinal cells pumping cholesterol back to the lumen
As cell debris derived from the rapid turnover of intestinal cells.
Mostly it is absorbed in the duodenum and proximal jejenum. In humans 30-50% of cholestrol in the lumen is absorbed and returned to the liver, while the rest is eliminated with the feces.
Dietary cholesterol mixes with the biliary cholesterol and offered to the brush border of the small intestine in the form of mixed micelles. Transportation of cholesterol across the plasma membrane of the enterocyte through a pump recently identified as the niemann-Pick C1 like1protein (NPC1L1). A fraction of this cholesterol is pumped back into the intestinal lumen by ATP-binding cassettes hemi transporters ABCG5 and ABCG8,while the remaining moves to the endoplasmic reticulum where it is esterified by the enzyme acyl-coenzyme A. Cholesterol acyl transferase 2 (ACAT2) and incorporated into nascent lipoproteins and the NPC1L1 sterol transporter has 50% homology with NPC1, which is involved in intracellular chlesterol trafficking and storage.
ABCG5 and ABCG8 actively transports cholesterol in the intestine . They are also expressed in the liver where they serve as pumps for the cholesterol secretion of ABCG5 into bile. ABCG8 and ABCG5 each encode a protein with 6 trans membrane domains, therefore a dimerization to form a 12- transmembrane protein complex is required for transport activities.
Mutations in either hemitransporter cause sitosterolemia, a condition characterized by over absorption of plant sterols and dietary cholesterol.
Affected people absorb 15-20% of plant sterols instead of normal 1-3%. Biliary sterol excretion will be reduced. Plant sterols enter the enterocyte but, they are poor substrates for ACAT2, they remain unesterified until they pump back to the intestinal lumen by ABCG5/G8. Plant sterols may be atherogenic compounds that the body efficiently expels as general defence mechanism.
Bile acid binding resins (cholesteramine) or selective lipase inhibitors (Orlistat) reduce cholesterol absorption by interfering with the processes other than cholesterol transporters. ACAT2 and partial CETP inhibitors are currently used in clinical trials for the same purpose.
Diets with high amount of fats or cholesterol causes both hyper-triglycedemia and hyper-cholesterolemia which are foremost scenario for cardiovascular diseases and primary causes of death in countries which are developing and developed. Estimation of world health organization (WHO) is that by 2010 60% of the world's heart patients will be Indians.
1.8 Cholesterol esterase
Dietary cholesterol comprised of free and esterified cholesterol.In diets rich in meats,significant percent of cholesterol is esterified. Hydrolysis of cholesteryl ester in the lumen is catalysed by cholesterol esterase
( CEase).
Cholesterol esterase is an acid lipase ,which is synthesized in the pancreas, catalyses the hydrolytic cleavage of cholesterol,sterol esters and triglycerides.
The name pancreatic cholesterol esterase is ascribed to the only enzyme in the pancreas that hydrolyzes cholesterol esters to unesterified cholesterol and free fatty acids. However, extensive investigations over a period of more than 30 years revealed that a protein with similar properties can also be purified from homogenates of several other tissues and body fluids and that enzyme is a nonspecific lipase capable of hydrolyzing cholesteryl esters, vitamin esters, triacylglycerol, phospholipids, and lysophospholipids. At the onset of these investigations, it was not clear whether these various enzyme activities were properties of the same protein. Thus, this enzyme was also named nonspecific lipase, phospholipase A1 lysophospholipase, bile-salt-stimulated lipase, bile salt-dependent lipase, carboxyl ester lipase, and carboxyl ester hydrolase.
Sequence comparison with other proteins also revealed that this enzyme is responsible for the lipoamidase activity in milk, which may account for its ability to hydrolyse the physiological lipoamide substrate ceramide.
Nomenclature of this enzyme was made based on the various substrates of this enzyme.
Most commonly used name Carboxy ester lipase(CEL), based on the general reactivity of with lipids containing carboxyl ester bonds.
Cholesterol esterase or cholesterol ester lipase, due to its documented physiological function as acholesteryl ester hydrolase.
Bile-salt-stimulated or bile salt dependent lipase, based on unique bile salt dependency.
Cholesterol esterase has received most attention as having a potential role in cholesterol absorption. CEase has a broad substrate specificity, hydrolyzing tri-, di- and mono glycerides and phospholipids in vitro. It also hydrolyzes cholesterol esters, which form a part of dietary cholesterol and cannot be engrossed without prior hydrolysis to free cholesterol. As such, it is one of the essential enzymes that mediates absorption of dietary lipids through the intestinal wall into the blood stream. A number of studies have recommended a possible role for CEase in the absorption of free cholesterol at the brush border membrane of the small intestine , through a CEase gene .
1.9 Synthesis of cholesterol esterase (CEase)
Major tissues for the synthesis of this enzyme was acinar cells of exocrine pancreas and lactating mammary glands.
Enzyme synthesized bythe pancreas is hoard in zymogen granules and is secreted with the pancreatic juice in a process stimulated by the gastric hormones such as cholecytokinin, secretin and bombesin. CEase mixes with the bile salt in the lumen of digestive tract and becomes active enzyme which catalyzes nutrient digestion and absorption through GIT.
Enzyme produced from the lactating mammary glands secreted as a major constituent of milk proteins and reaches the digestive tract of the infants, which plays a role in nutrient digestion and absorption in them.
Low but significant levels are also synthesized in other tissues like liver, eosinophils, endothelial cells and macrophages. Physiological function of this enzyme synthesized outside digestive tract is unknown.
1.9 Structure - Function Relationship
Primary structure of CEase which is deduced from the nucleotide sequencing of its cDNA from various species, indicates that this enzyme is highly conserved and is a member of the α/β-hydrolase. CEase utilizes a catalytic triad of Ser-His-Asp/Glu to form the charge relay network required for substrate hydrolysis. Site specific mutagenesis experiments documented that Ser194 is the key residue in CEase responsible for nucleophilic attack on the substrate carboxy ester bond. The reaction is assisted by His435 through a general catalysis of acid-base reaction on the carbonyl substrate. These also revealed the participation of Asp320 sin the catalytic triad serves by providing a better reaction of acid-base through pKa modulation.
CEase enzyme is active alone in hydrolyzing carboxyl esters containing short chain fatty acids, but it requires bile salt activation for the hydrolysis of carboxyl esters having fatty acyl groups of long chain. Numerous studies suggested the mechanism for bile salt activation of CEase , bile salt interacts with two sites on the protein which produce different effects.
One site is termed as nonspecific site , which has the capability to bind both di- and tri hydroxylated bile salts. Negatively charged side chain of bile salt interacts with one or more arginine residues in the enzyme. This protect CEase from proteolysis and promotes the binding of CEase to the surface of lipid emulsion prior to its hydrolysis of emulsified substrates. Bile salts binding to this site has no effect on CEase hydrolysis of water soluble substrates.
Second site is specific for trihydroxylated bile salts. Binding of the bile salts like cholate and taurocholate to this site induces a conformational change in the enzyme and increases hydrolytic activity against both water soluble and lipid soluble emulsified substrates.
The size of CEase protein differs from various species primarily due to the number of proline -rich repeating sequences near the carboxyl terminus of the protein.the largest CEase protein is the human enzyme at 100 kDa, which contain 16 repeating units with consensus sequence of PVPPTDDSQ. The rat , mouse, bovine and rabbit enzymes are smaller proteins at respectively 74 kDa and contain four, three and two such repeating units. These proline rich repeating units is important for maintaining the stability of the protein and these are the sites of
O-glycosylation. the carboxyl terminus of CEase also contain a domain that is required for normal intracellular processing and secretion of protein. The truncated CEase without any proline rich repeating units or the C-terminal domain also retain hydrolytic activities against both water soluble and lipid substrates, this suggests that repeating units donot participate in the catalytic activity of the protein. Modified enzymes without the C-terminal domain and with deletion of all proline rich repeating units were more active than native enzymes at low bile salt concentration in subatrate hydrolysis. This data says that C-terminal domain and proline rich repeating units are important in regulating the substrate accessibility to the active site of the protein.(chen et al.,)
According to the recent reports from the two different laboratories of X-ray crystal structure of bovine CEase provide additional support for the importance of the C-terminal domain in regulating substrate delivery to the active site domain of CEase. It is a glycoprotein of 579 aminoacids, with a notable proline-rich region between amino acids 540 and 573, and a highly conserved six-amino acid hydrophobic sequence forming the extreme C terminus of the protein. Crystallized truncated version of bovine CEase without the C-terminal repeats with 13β- strands and 14α-helices at .28nm resolution. Others made a contrast experiments and reported a structure with11β -strands and 15α-helices at .16 nm resolution using a full length protein without N-linked glycosylation. But both structures are similar in having the central location of the active site triad same as that noted in other lipases and esterases. Both crystal structures which are predicted from the molecular modelling of the protein indicates that CEase lacks the amphipathic helical lid domain of other lipases prominent for interfacial activation. But it contains atruncated lid with a pair of antiparellel β-strands, overlapping the N-terminal disulfide loopat residue 64-80.
1.10 Cholesterol absorption
Role of CEase in adult pancreatic secretion, has been debated but this was centered around the role of this enzyme in the absorption and esterification of dietary cholesterol. Hydrolysis of the cholesterol esters is the primary function of this enzyme. This function will not play a major role in cholesterol absorption because cholesteryl esters represent only 10% of dietary cholesterol. The ability of CEase to re-esterifycholesterol in vitro and its localization within the interstitial cells in immunohistochemistry studies led to the hypothesis that CEase may either serve as docking or a carrier protein for free cholesterol uptake by enterocytes. It had been postulated that CEase mediates the re- esterification of cholesterol after it transverse through the membrane bilayer and this step is required prior to the cholesterol secretion into the lymph as part of chylomicrons and very low density lipoproteins.
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Role of cholesterol esterase in cholesterol absorption
1.11 Vitamin absorption
A natural CEase substrate present in the diet is vitamin A, which is present in the form of long chain fatty acyl esters of retinol. This absorption in natural form requires the hydrolysis of the ester in the similar way as that of cholesteryl ester absorption. Because CEase is able to recognize the retinyl esters as a substrate. So, It has been proposed that this enzyme may be important for the absorption of vitamin A.
Based on the evidence of the invivo studies done by using the rats which are fed with the tetrahydrolipstatin showed the reduced retinyl palmitate absorption.
1.12 Triglyceride absorption
Adequately triglyceride hydrolysis in adults was accomplished by the high concentration of pancreatic lipase. End products of this absorption are free fatty acids and monoglycerol both are efficiently absorbed by the small intestine. CEase may enhance the digestion and absorption by hydrolysing the monoacyl glycerol, But this physiological implication is not clear.
It was proved in some in vitro studies that CEase significantly enhanced the rate and extent of pancreatic lipase-catalysed triglycerides hydrolysis having long chain poly unsaturated fatty acids, such as arachidonate, eicosapentaenoate and docosahexaenoate. In the absence of CEase,pancreatic lipase was unable to digest 1,2-diacyl glycerol containing these fatty acid absorption. Thus CEase may play arole in poly unsaturated fatty acid absorption. This has to be tested in in vivo studies by using CEase knock out mice.
1.14 Phospholipid digestion
Physiological importance of the phospholipase A1 and lyophospholipase activities of CEase has not been addressed in literature. Thus this process is poorly understood. This process is mediated by the tye 1 phospholipase A2 secreted from the pancreas or phospholipase B synthesized by small intestine.the products generated from these enzyme activities, lysophospholipids and freefatty acids are efficiently taken up by enterocytes. Thus, it is unlikely that CEase plays any significant role in phospholipid absorption.
One exception to this hypothesis is that the cod lacks both the enzymes that are useful in phospholipid digetion . thus CEase is the only lipolytic enzyme in the intestinal lumen of the cod and may function in lipd absorption in this species. Hence CEase in other species may also serve a backup function to other lipolytic enzymes for transport and uptake of these vital nutrients.
1.15 Neonatal nutrition
Considerable amount of direct and indirect evidence has accumulated to support the hypothesis that milk- derived CEase plays an important role in neonatal nutrition, especially in the digestion and absorption of milk fat and fat-soluble vitamins . comparision between very low birth weight infants fed either raw, pasteurized, or boiled human milk. Fecal fat output doubled when the infants were fed heat-treated milk and their fat absorption rates were decreased by 30% to 40%. It was found that excretion of free fatty acids in the fecal fat was significantly greater after feeding the infants with raw milk, including that malabsorption was a reflection of reduced digestion of the milk fat. Weight gain was greater during the period the infants were being fed the raw milk. These findings suggested that a heat labile factor in the milk was important for proper digestion and absorption of fat by preterm infants. Alternate explanation for this is that heat treatment may alter the structure of the milk globule so that it is less digestible by either the gastric or the panceatic lipase.
