Lipids and lipoproteins

Cholesterol absorption

Cholesterol enters the intestinal lumen from three sources: the diet, bile, and the intestine. Animal products—especially meat, egg yolk, seafood, and dairy products—provide the bulk of dietary cholesterol. Although dietary cholesterol intake varies considerably, the average American diet contains approximately 300 to 450 mg of cholesterol per day and 200 to 250 mg of phytosterols. ^, A much larger amount of cholesterol enters the gut from biliary secretion (3 to 10 times higher than dietary sources). Additional quantities arise from the turnover of intestinal mucosal cells (∼300 mg) and from direct intestinal secretion. Practically all cholesterol in the intestinal lumen is present in the unesterified or free form. Esterified cholesterol, which accounts for approximately 15 to 20% of dietary cholesterol, is rapidly hydrolyzed in the lumen of the intestine to free cholesterol and free fatty acids by cholesterol esterases secreted from the pancreas and small intestine. Because the majority of the intestinal pool of cholesterol is from endogenous rather than exogenous sources, there is not a close relationship between dietary cholesterol intake and coronary atherosclerosis. Consistent with this observation, since 2013 the American Heart Association (AHA) and American College of Cardiology (ACC) guidelines on the treatment of blood cholesterol have stated that there is insufficient evidence that lowering dietary cholesterol has a major impact on plasma cholesterol levels.

To be absorbed, unesterified cholesterol must first be solubilized by emulsification. This occurs through the formation of mixed micelles that contain unesterified cholesterol, phytosterols, stanols (saturated sterols), fatty acids, monoacylglycerides (derived from dietary TG), lysophospholipids, and conjugated bile acids. Formation of mixed micelles promotes cholesterol absorption in the brush border of the proximal small intestine by both solubilizing cholesterol and facilitating its transport to the surface of the luminal cell, where it is absorbed by an active process involving an enterocyte membrane sterol influx protein called Niemann Pick C1 Like 1 protein (NPC1L1). Loss of function of the NPC1L1 gene is associated with both reduced plasma cholesterol concentrations and reduced risk of CHD. ^, NPC1L1, which is also expressed at the hepatobiliary border, is the drug target for the cholesterol absorption inhibitor ezetimibe.12–14 Because of their strong detergent-like effects, bile acids are the most important factor in micelle formation. In the absence of bile acids, digestion and absorption of both cholesterol and TG are severely impaired, leading to fat malabsorption. In healthy individuals, the degree of cholesterol absorption can vary widely, but on average about 50% of intestinal cholesterol is absorbed while the remainder leaves the body via stools.

Absorption of cholesterol and phytosterols is limited by the presence of a sterol efflux transporter called the ATP binding cassette (ABC) transporter G5/G8, which is a heterodimer of G5 and G8. The ABCG5/G8 transporter is located at the luminal and biliary borders of enterocytes and hepatocytes, respectively, and facilitates sterol and stanol efflux from the body back into the gut lumen or biliary tree for return to the gut. Polymorphisms that cause partial loss of function of ABCG5 or ABCG8 result in hyperabsorption of cholesterol and mild to moderate degrees of phytosterolemia. Total loss of function of either ABCG5 or ABCG8 results in an autosomal recessive familial sitosterolemia, also termed phytosterolemia or xenosterolemia . It is characterized by a marked increase in plasma and tissue concentrations of cholesterol and phytosterols, such as sitosterol, campesterol, and stigmasterol, and an increased risk of cardiovascular disease (CVD).

The ability of cholesterol to form micelles is also influenced by the quantity of dietary fat but not by its degree of saturation. Increased amounts of fat in the diet results in expansion of mixed micelles, which in turn allows more cholesterol to be solubilized and absorbed. As lipid absorption occurs in the small intestine, the micelles eventually break up, and the bile acids are either reabsorbed at the ileum or excreted. Any cholesterol not absorbed is excreted either as free cholesterol or as coprostanol or cholestanol after conversion by gut microbes.

After its absorption into enterocytes, cholesterol and phytosterols have several possible fates. Acyl-coenzyme A:cholesterol acyltransferases (ACAT) may esterify cholesterol to cholesteryl ester (CE). Free sterols can also be effluxed or pumped out of enterocytes by the ATP binding cassette transporters A1 (ABCA1) onto small HDL particles. In fact, about a third of plasma HDL cholesterol is formed by the gut during this process. In addition to acting as a lipid absorption organ, the intestine can also act as a secretory organ by returning excess esterified cholesterol, stanols, phytosterols, and free cholesterol back to the gut lumen via ABCG5/G8 transporters. Because of the combined actions of NPC1L1 and ABCG5/G8 transporters, relatively small amounts of phytosterols and stanols ever reach the systemic circulation. This allows absolute concentrations of phytosterols and stanols or their ratios to TC to be used as biomarkers of cholesterol absorption. ^,

Lipids, including TGs, phospholipids (PL), free and unesterified sterols, and a number of specific apolipoproteins, with the help of microsomal transfer protein (MTP) and apolipoprotein B-48 (apoB-48), are assembled into large lipoproteins called chylomicrons. Patients with a rare deficiency in this process develop a disease called chylomicron retention disorder , which is characterized by excessive lipid accumulation in enterocytes and fat malabsorption. Chylomicrons are secreted into the lymphatics and eventually enter the thoracic duct, which connects to the systemic venous circulation at the junction of the left subclavian vein and the left internal jugular vein.

Cholesterol synthesis

In addition to dietary sources, cholesterol can be synthesized by all tissues from acetyl-CoA ( Fig. 36.2 ). Knowledge of this biochemical pathway, which took decades to elucidate, has acquired great significance, because the most commonly used drug agents for lowering plasma cholesterol (statins) act on the rate-limiting step in this pathway—namely, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase. Bempedoic acid is another cholesterol-lowering drug that inhibits adenosine triphosphate-citrate lyase (ACL), an enzyme upstream of HMG-CoA reductase. The necessity for understanding the fundamental biochemistry of this pathway was originally underscored by the triparanol disaster of 1960. Triparanol is a drug that inhibits the final step in the endogenous cholesterol synthetic pathway—the conversion of desmosterol to cholesterol—but it does not inhibit HMG-CoA reductase (see Fig. 36.2 ). When triparanol was used to treat hypercholesterolemia, the drug caused tissue accumulation of desmosterol, resulting in the development of cataracts, alopecia, and accelerated atherosclerosis.

Although both the liver and the small intestine play a major regulatory role in cholesterol homeostasis, all cells have the capacity to synthesize cholesterol from acetate. Extrahepatic tissues are responsible for greater than 80% of TC production. Cholesterol biosynthesis can be conceptualized as occurring in three stages (see Fig. 36.2 through Fig. 36.4 ). In the first stage, acetyl-CoA, a key metabolic intermediate that can be derived from carbohydrates, amino acids, and fatty acids, forms the six-carbon thioester HMG-CoA. In the second stage, HMG-CoA is reduced by HMG-CoA reductase to mevalonate, which is then decarboxylated to form a five-carbon isoprene structure. These isoprenes are condensed to form first a 10-carbon (geranyl pyrophosphate) and then a 15-carbon intermediate (farnesyl pyrophosphate). Two of these C ₁₅ molecules combine via the enzyme squalene synthetase to form the final product of the second stage: squalene, a 30-carbon acyclic hydrocarbon. The second stage is important because it contains the regulatory enzyme HMG-CoA reductase, as well as the enzyme geranyl transferase, the second important site of cholesterol regulation. At this step, there is the regulated diversion of farnesyl pyrophosphate from the synthesis of cholesterol for the production of other physiologic lipids, such as dolichol or the modification (prenylation) of important membrane anchored proteins, such as Ras with farnesyl or geranylgeraniol groups.

The third and final stage of cholesterol synthesis occurs in the endoplasmic reticulum, with many of the lipid intermediate products being bound to a specific carrier protein. Squalene, after initial oxidation, undergoes cyclization to form lanosterol, a four-ring, 30-carbon intermediate. Lanosterol then undergoes several transitions either by the Kandutsch-Russell or Bloch pathways to become cholesterol with lathosterol and desmosterol, respectively, as the penultimate intermediates. The enzyme that dictates which pathway to be used is determined by the stage at which the double bond at position C24 of the aliphatic side chain is reduced. Defects of enzymes in these pathways can lead to desmosterolosis, lathosterolosis, Smith-Lemli-Opitz, and other malformation syndromes. The most common biomarkers used to assess cholesterol synthesis are absolute concentrations of desmosterol and lathosterol or their ratios to TC.

Cholesterol esterification

The majority of cholesterol in the body is stored in cells or is transported in lipoprotein cores as hydrophobic CE molecules. The fatty acids most frequently esterified to the hydroxyl group of cholesterol are the 16-carbon unsaturated palmitic acid or the 18-carbon monounsaturated oleic acid, creating cholesteryl palmitate or oleate, respectively. Several intracellular enzymes (esterases) exist that can convert CE back to free cholesterol.

Almost all of the cholesterol in plasma is bound to lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), LDL, Lp(a), or HDL. The major apolipoprotein found in VLDL, IDL, LDL, and Lp(a) is apoB-100, a large protein that contains over 4500 amino acids. The apoB-48 protein is a truncated version of apoB-100 found on chylomicrons and chylomicron remnants. It is produced in the intestine by a post-transcriptional editing step, which introduces a stop codon in the middle of the apo B-100 messenger ribonucleic acid (mRNA) transcript, thus resulting in a protein that is about 48% the length of the full-length apoB-100 protein.

The esterification of cholesterol is critical because it serves to enhance the lipid-carrying capacity of lipoproteins and prevents intracellular toxicity by free cholesterol. In the plasma, the reaction is catalyzed by lecithin-cholesterol acyltransferase (LCAT) and in cells by acylcholesterol acyltransferase (ACAT). ACAT is an energy-requiring enzyme, and the initial reaction ( Fig. 36.5 ) involves activation of a fatty acid with thio-coenzyme A (CoASH) to form an acyl-CoA, which in turn reacts with cholesterol to form CE. In contrast, the LCAT reaction does not require CoASH and transfers a fatty acid from the second carbon position of phosphatidylcholine (lecithin) to the hydroxyl group on the A-ring of cholesterol. CEs account for about 70% of the TC in plasma, and LCAT is responsible for the formation of most, with the residual being produced by ACAT that was released into the circulation during the secretion of lipoproteins. Two different LCAT activities have been described, with α-LCAT esterifying the free cholesterol of HDL and β-LCAT activity occurring on apoB-containing lipoproteins. LCAT is synthesized in the liver and released into the circulation; it primarily resides on HDL and to a lesser degree on LDL and is activated by apoA-I. The esterification of cholesterol by LCAT on the polar hydroxyl group of cholesterol makes CE more hydrophobic than cholesterol. This esterified cholesterol partitions into the more hydrophobic core of lipoproteins, where TGs are also stored. This is important in the maturation or enlargement of HDL particles, allowing the surface to accommodate more free cholesterol from cellular efflux.

Cholesterol catabolism

Once a lipoprotein enters a cell, its CEs and glycerol esters (most importantly TGs and PL) are hydrolyzed in lysosomes by lysosomal acid lipase (LAL), which is encoded by the gene LIPA. Partial or complete lack of this enzyme results in a lysosomal storage disorder, resulting in the intracellular accumulation of CEs and TGs, particularly in the liver. The partial loss of LAL results in the late-onset form of the disease and produces a clinical disorder known as cholesteryl ester storage disease (CESD). CESD should be suspected in adults with dyslipidemia associated with elevated transaminases and LDL-C, and with reduced HDL-C. Unlike other forms of dyslipidemia, less than 50% of patients with this disease have increased plasma TG levels. The almost complete loss of activity of this enzyme presents usually shortly after birth as Wolman disease , which often results in liver failure from lipid accumulation. A recombinant human LAL known as Sebelipase alfa is approved as a therapy.

Cholesterol reaching the liver may be secreted unchanged into bile as free cholesterol, metabolized to bile acids, or incorporated into and secreted back into the circulation on lipoproteins. Approximately one-third of the daily production of cholesterol (about 400 mg/day) is converted into bile acids ( Fig. 36.6 ). Conversion of cholesterol to cholic and chenodeoxycholic acids, the major bile acids in humans, involves shortening of the cholesterol sidechain and hydroxylation of the sterol nucleus. The first step, which is also the rate-limiting step, is hydroxylation of the 7-position, catalyzed by the enzyme 7α-hydroxylase. The bile acids are made even more polar after conjugation with glycine or taurine and then are excreted into the bile, where they play an active role in fat absorption, as discussed previously. Some of the bile acids are deconjugated by bacteria and converted into secondary bile acids. Cholic acid is converted to deoxycholic acid, and chenodeoxycholic acid is metabolized to lithocholic acid. Except for lithocholic acid, about 90% of the bile acids are reabsorbed in the lower third of the ileum and returned to the liver in the portal vein by the enterohepatic pathway (see also Chapter 51 ).

A significant amount of cholesterol and phytosterols are also excreted from enterocytes and hepatocytes via the ABCG5/G8 transporter back into the gut lumen or bile, where they are resolubilized with bile salts and PL. If the amount of cholesterol in bile exceeds the capacity of these solubilizing agents, the excess cholesterol can precipitate, forming cholesterol gallstones, which account for about 80% of gallstones in Western societies. It is important to note that except for the liver and a few endocrine tissues (adrenal glands and gonads), most cells cannot further catabolize or modify cholesterol. Because of this and its limited aqueous solubility, cholesterol tends to accumulate, triggering cellular apoptosis or forming extracellular crystals, both of which can contribute to the development of atherosclerosis. As discussed below, cells have the ability to rid themselves of excess cholesterol to HDL via active sterol efflux pumps or by free diffusion to lipoproteins, erythrocytes, or albumin.

Fatty acid catabolism

Long-chain fatty acids are oxidized in the mitochondria and produce cellular energy by a series of reactions that operate in a repetitive manner to sequentially shorten the fatty acid chain by two carbon atoms at a time from the carboxy (—COOH) terminus in a process known as β-oxidation. For example, 1 mole of C ₁₆ fatty acid is converted to 8 moles of acetyl-CoA. Acetyl-CoA does not accumulate in the cell but is enzymatically condensed with oxaloacetate, derived largely from carbohydrate metabolism ( Fig. 36.8 ), to yield citrate, a major component of the tricarboxylic acid cycle or Krebs cycle. The Krebs cycle serves as a common pathway for the final oxidation of nearly all food material, whether derived from carbohydrate, fat, or protein. It is important to note that the efficiency of the Krebs cycle depends on the availability of sufficient oxaloacetate to serve as an acceptor for acetyl-CoA.

Complete oxidation of a single fatty acid molecule produces a relatively large quantity of energy. For example, the complete oxidation of 1 mole of palmitic acid produces 16 moles of CO ₂ , 16 moles of H ₂ O, and 129 moles of adenosine triphosphate (ATP), or 2340 Calories (Cal). The unit used in discussing the energy value of food is the Calorie (Cal), equal to 1000 calories or 4.19 joules. Thus the standard free energy for oxidation of palmitic acid is 2340 Cal, whereas the free energy liberated by hydrolysis of 129 moles of ATP is only 940 Cal, indicating that the efficiency of energy conservation in fatty acid oxidation is approximately 40% under standard conditions.

By means of suitable enzyme reactions, the chemical energy stored in fatty acids can be released for metabolic processes or stored in the form of high-energy compounds, such as ATP or creatine phosphate. TGs are an efficient storage form for metabolic energy. The amount of energy produced by metabolizing 1 mole of palmitic acid (16 carbon atoms) is approximately twice that produced by metabolizing a similar mass (2.5 mole) of glucose (6 carbon atoms per molecule). Carbohydrate storage also requires a lot of water for hydration; TG storage does not. In addition to their high intrinsic energy content, the storage of TGs in subcutaneous fat deposits provides insulation for the body.

Ketone formation

During prolonged starvation, or whenever carbohydrate metabolism is severely impaired, as in untreated type 1 diabetes mellitus (see Chapter 47 ) or intentionally induced nutritional ketosis from carbohydrate restriction, the formation of acetyl-CoA exceeds the supply of oxaloacetate. The abundance of acetyl-CoA results from excessive mobilization of fatty acids from adipose tissue and their conversion by β-oxidation in the liver. The resulting excess acetyl-CoA is diverted to an alternative pathway in the mitochondria to form acetoacetic acid, β-hydroxybutyric acid, and acetone—three compounds known collectively as ketone bodies . Increased ketone bodies are a frequent finding in uncontrolled type 1 diabetes mellitus, but much lower levels can also result from nutritionally induced ketosis. As shown in Fig. 36.9 , the first product, acetoacetyl-CoA, condenses in the mitochondria with a third molecule of acetyl-CoA to yield HMG-CoA. This pool of HMG-CoA in the mitochondria is distinct from the pool in the cytosol, which is used for cholesterol biosynthesis. The HMG-CoA produced in the mitochondria is cleaved enzymatically to yield acetoacetate and acetyl-CoA. Some of the acetoacetate formed in liver cells is reduced to β-hydroxybutyrate. Because acetoacetate is unstable, a further portion decomposes to form carbon dioxide and acetone, the third ketone body found in high concentrations in pathologic ketotic states. Ketosis, therefore develops from excessive production of acetyl-CoA because the body attempts to derive energy from stored fat in the absence of an adequate supply of carbohydrate metabolites (see Chapter 35 ). In nutritional ketosis, the ketones serve as a physiologic energy supply and have been reported to increase LDL-C.

Inadequate incorporation of acetyl-CoA into the Krebs cycle may be further aggravated by inhibition of the oxaloacetate-generating enzyme system through excess accumulation of palmitic-CoA and other long-chain fatty acid–CoA derivatives in the liver. Skeletal muscle and the heart (and the brain in prolonged fasting) use ketone bodies by resynthesizing their CoA derivatives and subsequently oxidizing them for the production of energy. Although liver cells are largely responsible for generating ketones, they cannot metabolize acetoacetate because the liver lacks 3-ketoacid CoA transferase, the enzyme required for transferring CoA from succinyl-CoA.

The entire process of pathologic ketosis is reversed by restoring adequate metabolism of carbohydrate. In starvation, restoration consists of adequate carbohydrate ingestion. In diabetic ketoacidosis (DKA), ketosis can be reversed by insulin administration, which permits circulating blood glucose to be taken up by the cells. With restored concentrations of oxaloacetate, acetyl-CoA can then enter the Krebs cycle, thus restoring the normal pathway for energy metabolism. Eventually, the release of fatty acids from adipose tissue slows down and is finally reversed. A graphic view of these metabolic reactions is outlined in Fig. 36.8 , which shows the overall interrelationship between carbohydrate, fatty acid, and protein metabolism.

Prostaglandins

Prostaglandins and related compounds are derivatives of long chain fatty acids, such as arachidonate. This group consists of prostaglandins, thromboxanes, some hydroperoxy—and hydroxy—fatty acid derivatives, and leukotrienes. There remains a paucity of widespread clinical application for prostaglandin diagnostics at this time.

Glycerophospholipids

Glycerophospholipids (PL) are the main component of cell membranes, as well as the surface of lipoproteins keeping hydrophobic TGs and cholesterol esters in solution in the water phase of plasma. ^, PLs contain phosphoric acid at the third (α′) carbon atom ( Fig. 36.11 ). In their simplest form, the A group is an H atom, so the molecule is called a glycerophosphate . Usually, however, the A is an alcohol-derived group, such as choline, serine, inositol, or ethanolamine (see Fig. 36.11 ). If A is choline, the molecule is referred to as phosphatidylcholine; if it is ethanolamine, it is referred to as phosphatidylethanolamine; and so on. The term lecithin, which is an older designation, is still commonly used for phosphatidylcholines. Because of the wide variety of fatty acid residues at positions R ₁ and R ₂ (see Fig. 36.11 ), many different types of PL can be formed. These PL are named according to the fatty acid acyl ester attached at C-1 and C-2 of the glycerol. Saturated fatty acids are typically attached to the C-1 position, whereas (poly)unsaturated fatty acids are often present at the C-2 position. A PL that has lost one of its O-acyl groups is called a lysophospholipid. In inner mitochondrial membranes, more complex phosphoglycerides known as cardiolipins can be found. They are derived from two phosphoglyceride molecules joined by a glycerol bridge. Enzymes that hydrolyze PL are termed phospholipases or lysophospholipases .

Apolipoprotein a family

Most apolipoproteins, including those in the apoA family, contain a structural motif called an amphipathic helix. It is an α-helix with approximately half the amino acid residues comprising hydrophobic amino acids, which face toward the neutral lipid core when bound to a lipoprotein particle. The other side of the helix faces outward toward the surface of a lipoprotein particle and contains polar or charged amino acids. In general, the binding of amphipathic helices to lipoproteins is relatively weak, thus allowing apolipoproteins (except apo(a) and apoB) to readily exchange between different lipoproteins during their metabolism.

Together, apoA-I and apoA-II constitute about 90% of total HDL protein. The ratio of apoA-I to A-II in HDL is about 3:1. In addition to being an important structural component of HDL, apoA-I is a ligand for the major cellular membrane sterol efflux protein, ABCA1. It is also a cofactor for LCAT, the enzyme responsible for esterifying free cholesterol, a crucial step in the maturation and remodeling of HDL. ApoA-I can be present from one to five copies per HDL particle, and it is the degree of twisting of apoA-I around the HDL particle surface that modulates particle size. ApoA-I on spherical HDL particles has been proposed to exist in a trefoil configuration when three copies are present, but it can accommodate more copies in a similar structural arrangement.

The exact role of apoA-II is unclear, but there is some evidence that it inhibits hepatic lipase (HL). ApoA-II can also delay the lipolysis of large TG-rich lipoproteins by interfering with lipoprotein lipase (LPL). ApoA-IV, which is found in the apoA-I/C-III/A-IV gene cluster on chromosome 11, is synthesized in the intestine and is secreted as a component of chylomicrons. Chylomicrons may contain a variable number of apoA-IV proteins, which may allow them to exist in a wide spectrum of sizes. ApoA-IV may also contribute to the lipolysis of lipoproteins by facilitating the release of apoC-II from either HDL or VLDL. Other potential functions of apoA-IV are activation of LCAT, promoting intestinal lipid absorption and satiety through a hypothalamic effect.

Another recently recognized apolipoprotein is apoA-V. It is relatively low in abundance compared with other apolipoproteins and appears to modulate TG concentrations by several mechanisms, including modulating VLDL secretion and enhancing LPL function. ApoA-V, as part of TG-rich lipoproteins, also traffics with the particle and binds to glycosylphosphatidylinositol-anchored HDL binding protein 1, thus facilitating its interaction with LPL. Several polymorphisms of apoA-V have been associated with hypertriglyceridemia.

Apolipoprotein B

As already discussed, apoB exists in two forms: apoB-100 and apoB-48. Most of the apoB in plasma is apoB-100. ApoB-100, a single polypeptide of more than 4500 amino acids, is the full-length translation product of the APOB gene. In humans, apoB-100 is made in the liver and is secreted into plasma as part of VLDL, IDL, Lp(a), or LDL. ApoB-100 is also the major apolipoprotein of LDL and its measurement can serve as a surrogate for LDL particle concentration (LDL-P) when TG-rich lipoproteins and Lp(a) are not elevated. Unlike other apolipoproteins, however, apoB-100 is not transferable and cannot move from one lipoprotein particle to another because in addition to amphipathic helices, it has β-sheets—a structural motif with much higher affinity for lipids. It is for this reason that apoB-100 remains bound with VLDL and its lipolytic products IDL and LDL.

ApoB-48 contains 2152 amino acids and is identical to the amino-terminal portion of apoB-100. ApoB-48 results from post-transcriptional modification of internal apoB-100 mRNA, in which a single base substitution produces a stop codon corresponding to residue 2153 of apoB-100. ApoB-48 is made in the intestine and is the major apoB component of chylomicrons. Both apoB-100 and apoB-48 play important roles in the secretion of VLDL and chylomicrons, respectively. ApoB-100 is recognized by the LDL receptor (LDLR) in hepatic and peripheral tissues; it allows LDLR mediated internalization of LDL. ApoC-III and apo(a) can camouflage the LDLR binding domain, hindering LDLR mediated clearance of apoC-III–containing particles and Lp(a), respectively. ^,

Apolipoprotein C family

The apoC family mainly consists of three closely related proteins—apoC-I, apoC-II, and apoC-III—that are mostly made by the liver and, to a lesser degree, in the intestine. Another member of this family—apoC-IV—does not appear to be present in significant amounts in human serum. ApoC-I, the smallest of the C apolipoproteins with 57 amino acids, has been reported to activate LCAT and is also known to inhibit LPL, HL, phospholipase A2, and CETP. In fact, it accounts for most of the CETP-inhibitory activity found in human plasma HDL. ApoC-II, consisting of 78 amino acids, plays an important role in the metabolism of TG-rich lipoproteins (VLDL and chylomicrons) by acting as an activator of LPL. ^, Because of differences in sialic acid content, apoC-III, a 79 amino acid glycoprotein, exists in at least three different isoforms termed 0, 1, and 2. ApoC-III ₁ and apoC-III ₂ correlate more strongly with TG levels than apoC-III ₀ . It is also associated with the generation of small LDL. Recent studies reveal that apoC-III stimulates VLDL assembly and secretion and interferes with VLDL receptor, LDL receptor–related protein (LRP), and LDLR uptake of lipoproteins but does not, as previously thought, decrease lipolysis by direct inhibition of LPL. In hypertriglyceridemia, most VLDL is secreted with apoC-III but without apoE, and such particles are not cleared until they lose apoC-III during lipolytic conversion to dense LDL. LDLs that contain apoC-III are reportedly associated with ASCVD. ^, ApoC-III is also implicated in several inflammatory pathways. Mendelian randomization studies have shown loss-of-function mutations in APOCIII to be linked to favorable lipid profiles with low TG-rich lipoproteins and lower incidence of coronary artery disease. Antisense therapy with oligonucleotides that interfere with apoC-III synthesis are investigated by clinical trials, and one such drug (Volanesorsen) has been approved in Europe to reduce TGs and risk of acute pancreatitis in individuals with the familial chylomicronemia syndrome (FCS).

Apolipoprotein E

ApoE is a 34-kDa plasma glycoprotein containing 299 amino acids. It is synthesized primarily by the liver but is also produced locally by many other tissues and cell types, such as in the brain and by macrophages. ApoE is found on all lipoproteins, but only a small amount is on LDL. Removal of apoE–bearing lipoproteins is mediated by several different cellular receptors that recognize a cluster of positively charged amino acids in a specific region of apoE. It regulates lipoprotein uptake in the liver through the interaction of a wide variety of receptors, such as the chylomicron remnant receptor, the LRP, and the LDLR. It also promotes the interaction of lipoproteins with proteoglycans.

Three common apoE isoforms, designated E ₂ , E ₃ , and E ₄ , can be separated by isoelectric-focusing electrophoresis. These isoforms have amino acid substitutions at residues 112 and 158. ApoE ₂ has cysteine residues in both positions, and apoE ₄ has arginine residues in both positions, whereas apoE ₃ has cysteine and arginine at positions 112 and 158, respectively. ApoE ₂ has reduced binding affinity for the B and/or E remnant receptor compared with apoE ₃ , which can result in the accumulation of apoE–containing lipoproteins in the circulation. In contrast, apoE ₄ –containing lipoproteins are cleared more rapidly than those containing apoE ₃ . These isoforms are coded for by three alleles of the apoE gene: ε2, ε3, and ε4. The ε3 allele is most frequent, although relative proportions of the three alleles vary among populations. These apoE alleles have been shown to contribute significantly to the variability of LDL-C and apoB concentrations within populations. Individuals with at least one ε2 allele tend to have lower concentrations of apoB and LDL-C than do those who are homozygous for the ε3 allele, whereas individuals with at least one ε4 allele tend to have higher concentrations of apoB and LDL cholesterol. This most likely occurs because increased hepatic uptake of lipoproteins in the presence of the ε4 allele leads to an increase in hepatic cholesterol and downregulation of LDLR. ApoE ₄ is also associated with increased cholesterol absorption. In the distant past, this may have offered a selective evolutionary advantage for humans on calorie-restricted and low-fat diets, but it now appears to be a disadvantage in regard to the development of atherosclerosis on our current high-fat diets. Statin hyporesponsiveness has often been noted in apoE4 carriers, which may be related to the lesser efficacy of statins in patients who hyperabsorb cholesterol. A meta-analysis of 24 trials, however, suggested there was little clinical utility for APOE genetic testing for guiding treatment with statins. Although the ε2 allele is a strong genetic determinant of low Lp(a) concentrations, it does not modify the causal association of Lp(a) with myocardial infarction or aortic valve stenosis. ^,

Epidemiologically, the apoE ₄ allele has been strongly associated with late onset Alzheimer disease and other neurologic diseases, but newer data suggest that risk is not only isoform dependent but also directly related to apoE concentration. This association is likely related to the role of apoE in modulating lipid metabolism in the brain, but the exact connection between apoE ₄ and neurologic disease is not known.

Intestinal (exogenous) pathway

The primary function of the intestinal pathway is the absorption of dietary lipids and delivery, particularly TG, to peripheral tissues and the liver. This pathway begins when nascent chylomicrons are assembled from dietary TG and cholesterol in the enterocytes and stored in secretory vesicles in the Golgi apparatus. Chylomicrons are then released by exocytosis into the extracellular space and enter the circulation by way of lymphatic ducts. The lipid content of nascent chylomicrons consists mainly of TG (90% by mass) and only a small amount of protein, mostly apoB-48 and various apoA isoforms (2% by mass). Shortly after secretion, these lipoprotein particles quickly acquire apoC-II and apoE from circulating HDL (see Fig. 36.15 ). ApoC-II on the surface of chylomicrons promotes lipolysis of TG by activation of LPL, which is mostly attached to the luminal surface of endothelial cells. The released free fatty acids generated by lipolysis associate with albumin and can be taken up by muscle cells as an energy source or by adipose cells for storage after conversion back to TG. Simultaneously, some of the PL on chylomicrons are transferred back to HDL during this process. The partially lipolyzed chylomicrons, called the chylomicron remnants, are smaller and contain 10 to 20% less TG than the original nascent chylomicron. Because of the presence of apoB-48 and apoE on their surface, chylomicron remnants are recognized by specific hepatic remnant receptors and are quickly internalized within hours by receptor-mediated endocytosis and are further hydrolyzed within the lysosomes. Proteoglycans in the hepatic sinusoids also contribute to the uptake of lipoproteins. Cholesterol that enters hepatocytes can be used in bile acid synthesis, incorporated into newly synthesized lipoproteins, effluxed to apoA-I particles, secreted directly into the bile, or stored as CE. Furthermore, cholesterol from chylomicron remnant uptake downregulates HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis.

With respect to cholesterol, the vast majority (85 to 90%) of cholesterol that enters the intestinal lumen is from endogenous, not dietary (exogenous), sources. Large amounts of endogenously produced cholesterol enter the gut lumen via hepatobiliary delivery: direct intestinal secretion routes, termed transintestinal cholesterol efflux (TICE), or enterocyte membrane shedding of cholesterol. After absorption, cholesterol is used in chylomicron formation or in the formation of nascent HDL by a process dependent on the ABCA1 transporter. Further complicating the issue about the source of cholesterol is that intestinally absorbed fatty acids of exogenous origin, which are first incorporated into enterocytes as TG in chylomicrons, start exchanging TG by CETP with other lipoproteins, immediately after entering the circulation. In this way the endogenously produced lipoproteins produced by the liver (VLDL, IDL, LDL, and HDL) rapidly acquire and traffic exogenous lipids as well.

Hepatic (endogenous) pathway

The hepatic pathway delivers lipids that are packaged in the liver to peripheral cells (see Fig. 36.16 ). As discussed previously, however, chylomicrons also deliver endogenously produced cholesterol, and much of the lipoprotein transport of lipids is not only delivered to peripheral cells but also back to the gut or liver. When dietary cholesterol acquired from the receptor-mediated uptake of chylomicron remnants is insufficient, hepatocytes can also synthesize their own cholesterol by increasing the activity of HMG-CoA reductase or acquire cholesterol via internalization of LDL particles or through the delipidation of HDL particles when it interacts with the scavenger receptor B1 (SR-BI). Endogenously made TG and acquired or synthesized cholesterol are packaged along with apoB-100 into VLDL particles in the endoplasmic reticulum of hepatocytes in a step involving the MTP. A total loss of function of the MTP results in the inability to secrete apoB–containing lipoproteins and leads to the condition called abetalipoproteinemia. VLDL is a TG-rich lipoprotein (55% by mass) that contains apoB-100 and variable amounts of apoE and apoC apolipoproteins. The liver may also directly secrete a small amount of IDL and LDL with or without apoE and apoC-III. Additional apoC apolipoproteins may be transferred from HDL to VLDL after it enters the circulation. Similar to chylomicron metabolism, apoC-II present on the surface of VLDL activates LPL on endothelial cells, which leads to the hydrolysis of VLDL TGs and the release of free fatty acids. During lipolysis, the particle decreases in size, and excess surface PL may be removed by PLTP and transferred to HDL. It is important to note, however, that the rate of hydrolysis of VLDL TG is significantly slower than that of chylomicron TG. The much larger chylomicrons have many more copies of apoC-II and apoE per particle than do VLDLs, thus enhancing their binding to LPL and clearance. The average residence time of TG in VLDL is 15 to 60 minutes, compared with only 5 to 10 minutes in chylomicrons.

During lipolytic catabolism of VLDL, as surface PL and core TG are hydrolyzed, and CETP mediates the exchange of core TG for CE with other lipoproteins, VLDL reduces in size allowing the apoCs and other apolipoproteins to be transferred to HDL, resulting in smaller CE-rich remnant VLDL. Then, VLDL remnants can be taken up by the liver or continue down the lipolytic cascade where they are converted to smaller, denser IDL particles, which can be removed by hepatic remnant receptors that recognize apoE. Alternatively, VLDL remnants can be removed from the circulation after interaction with hepatic proteoglycans, either by direct internalization or indirectly after transfer to hepatic remnant receptors. Both VLDL remnants and IDL contribute to the return of cholesterol to the liver in a process termed indirect reverse cholesterol transport . The lipolytic fates of VLDL and IDL are highly dependent on their content of apoC-III and E. As VLDL and IDL are depleted of core TG, excess surface components, such as PL, free cholesterol, and apolipoproteins, are transferred to existing HDL or are used in the generation of de novo HDL particles when they form complexes with lipid-free apoA-I.

CE molecules are also transferred from HDL to LDL by CETP in exchange for TG and this exchange can be inhibited by lipid transfer inhibitor protein or apolipoprotein F. This transfer of neutral lipids from apoA-I to apoB particles is termed heterotypic exchange in contrast to the homotypic exchange that occurs between different apoA-I particles or between different apoB particles. The net result of the coupled lipolysis and CE exchange reaction is the replacement of much of the TG core in the original VLDL with CE. In humans, about half of IDL is removed by the liver, and the other half undergoes further TG hydrolysis, leading to the generation of LDL. Most LDL and its cholesterol content are eventually returned to the liver or intestine by the LDLR or by non–receptor-mediated clearance. When LDL particles are present in excess, independent of size, they can infiltrate into the vessel wall, where their accumulation can contribute to the development of atherosclerosis.

Low-density lipoprotein receptor pathway

The mechanism by which LDL is removed from the circulation is reasonably well understood and primarily occurs via both LDLR and nonreceptor pathways. Compared with VLDL and chylomicrons, LDL has a relatively long residence time in the circulation of about 3 days. Specific receptors present on plasma membranes recognize and bind apoB-100 or apoE when present on LDL (see Figs. 36.16 and 36.17). LDL in the circulation can acquire a hepatic secreted protein called proprotein convertase subtilisin kexin type 9 (PCSK9). LDL particles (with or without PCSK9) bind to membrane-expressed LDLR via the LDLR binding domain on apoB-100 and then are internalized in clathrin-coated pits and fuse with endosomes, which are mediated by a protein called the LDL receptor adaptor protein 1 (LDLRAP1), also known as autosomal recessive hypercholesterolemia (ARH) clathrin adaptor protein. If PCSK9 is present on the complex, it directs the LDLR to a catabolic pathway, and the receptor is degraded. Without PCSK9, the LDL particles are catabolized, but the LDL-receptor protein is recycled back to the cell membrane, allowing for more efficient removal of LDL from the circulation. Once LDL is delivered to the lysosome, apoB-100 is degraded into small peptides and amino acids. CE is hydrolyzed to free cholesterol, making it available for the synthesis of cell membranes, steroid hormones in endocrine tissues, or bile acids in hepatocytes.

Cells have multiple pathways for regulating their cholesterol content, most likely because of the cytotoxicity of excess free cholesterol. Excess unesterified cholesterol (1) decreases the rate of endogenous cholesterol synthesis by inhibiting the rate-limiting enzyme HMG-CoA reductase; (2) increases the formation of CE from unesterified cholesterol, catalyzed by ACAT; and (3) inhibits the synthesis of new LDLR by suppressing transcription. Many different intracellular pathways are also available for coordinated gene regulation of cholesterol metabolism, but the sterol regulatory element-binding protein (SREBP) transcription factors, which sense intracellular cholesterol concentrations, appear to play the most central role.

Under normal circumstances, some LDL is taken up by extrahepatic tissues, mostly steroidogenic tissues and adipocytes, through LDLR, SR-B1, or non–receptor-mediated pinocytosis. Non–receptor-mediated uptake becomes important as plasma LDL concentrations increase, as in familial hypercholesterolemia (FH) when LDL penetrate from plasma across endothelial cells into the arterial intima. Non–receptor-mediated uptake is not saturable, is not regulated, and is probably largely due to the interaction of LDL with hepatic proteoglycans. Scavenger receptor A is also unregulated, and some recognize LDL that has been modified in various ways, such as oxidized LDL. Scavenger receptors A are largely found on macrophages, and this probably plays a role in the accumulation of lipid in the atherosclerotic plaque development. Macrophages that become engorged with CEs are called foam cells that are found in xanthomas and in atherosclerotic plaques.

High-density lipoprotein–mediated trafficking of cholesterol

The traditional concept of the reverse cholesterol transport (RCT) pathway has recently undergone radical rethinking and might be better described as HDL-mediated trafficking of cholesterol. Historically, RCT was thought to help the body maintain cholesterol homeostasis by removing excess cholesterol from peripheral cells and delivering it to the liver for excretion. The focus most often was on removal of cholesterol from the atherosclerotic plaque; however, the existence of such a process has never been documented in vivo in humans. It was believed to be mediated mostly by HDL, thus accounting for its suggested “antiatherogenic” property. However, recent evidence shows that this pathway is a much more complicated and dynamic process involving other lipoproteins, including LDL, multiple pools of cholesterol, the intestine, and other organ systems. Total RCT is the sum of direct and indirect pathways, which ultimately relocates or eliminates excess sterols from the body.

This pathway begins when lipid-poor apoA-I is secreted from the liver or the small intestine. This lipid-free apoA-I is named pre–β-HDL based on its electrophoretic migration. ApoA-I rapidly acquires PL and cholesterol from cells by the ATP binding cassette transporter 1 (ABCA1). ABCA1 is believed to pump excess cholesterol and other lipids to the outer surface of the plasma membrane, where apoA-I, in a detergent-like extraction process, removes PL and nonesterfied cholesterol and forms nascent HDL. The form of HDL produced in this process is discoidal in shape and forms a flat PL disc in a bilayer-like configuration because it is relatively depleted in neutral core lipids such as TG and CE (see Fig. 36.18 ). Two molecules of apoA-I stabilize nascent HDL by wrapping around the surface of the PL bilayer. Although the majority of HDL formed by this process occurs in the liver and intestine, ABCA1 is also present in peripheral cells, and it enables them to efflux excess cholesterol to HDL. This is believed to result in the generation of a larger discoidal species of HDL called α ₄ -HDL. Because the majority of cholesterol within HDL is of hepatic origin, the concept has recently emerged that HDLs traffic cholesterol in numerous directions and help the body equilibrate cholesterol among the various tissue pools beyond cholesterol back to the liver via RCT. HL and SR-BI are also believed to be responsible for regenerating smaller spherical forms of HDL and pre–β-HDL, respectively, from mature alpha _1-3 -HDL to restart this cycle.

As HDL acquires cholesterol, lecithin-cholesterol acyltransferase (LCAT-α) esterifies cholesterol by transferring fatty acids from the sn-2 position of neighboring PL, generating lysophospholipid and much more hydrophobic CE. CE then moves to the core of HDL, thereby transforming it from a discoidal to a spherical shape, which is the shape found in mature HDL. Lysolecithin is removed from the surface of lipoproteins by binding with albumin. The larger spherical forms of HDL, which are sometimes called α _1-3 -HDL based on their electrophoretic migration, can also acquire additional cholesterol by other cellular membrane transporters, such as by ABCG1 and the bidirectional sterol membrane CE transporter, SR-B1. As it matures, HDL can also acquire surface PL via PLTP, and smaller HDL particles can fuse, creating even larger species. Large HDL particles can also acquire unesterified cholesterol from cells via free diffusion or from other lipoproteins, erythrocyte membranes, or albumin-trafficked cholesterol. During this process, numerous (>100) serum proteins and other lipid moieties can also attach to various subsets of HDL particles, which may potentially contribute to their function.

Circulating cholesterol-rich, PL-rich, TG-poor HDLs have several options in dispensing their lipid cargo. CE can be transferred to other lipoproteins in exchange for TG via CETP. In this process, HDL particles can transfer CE to apoB–containing particles in a process called heterotypic exchange or to other HDL species in a process called homotypic exchange . Because they are by far the most numerous apoB–containing particles, much of the CE-TG exchange occurs between HDLs and LDLs. Potent CETP-inhibitors, which not only dramatically raise HDL-C but also significantly reduce LDL-C, suggest that a substantial number of the cholesterol molecules within LDLs are transferred from HDLs.

After receiving CE from HDL, LDL and other apoB–containing particles can traffic it to the liver or intestine in a pathway called indirect RCT. Additional options for HDL trafficking of cholesterol include direct delivery by SR-B1–mediated uptake by the liver, steroidogenic tissues, or adipocytes, which serve as cholesterol storage organs. HDL particles may also participate in direct RCT by other putative hepatic-located receptors, such as the holoparticle or mitochondrial-produced ATP synthase β-subunit, or by apoE receptor–mediated removal.

The liver has many options for “directly or indirectly” acquiring cholesterol: integrating it into the cell membranes, converting it to bile acids, lipidating it in newly forming VLDL, effluxing it to apoA-I, or directly excreting it to the biliary system via ATP binding cassette transporters G5 and G8 (ABCG5, ABCG8). The intestine can also promote cholesterol excretion or elimination by a new pathway called transintestinal cholesterol efflux (TICE). The exact pathway by which TICE promotes cholesterol excretion into the intestine is not known, but is thought to involve the direct transfer of cholesterol from either HDL or apoB–containing lipoproteins to the enterocyte, which then excretes it into the intestinal lumen.

As mature HDLs acquire TG via CETP exchange, they are subject to increased lipolysis by HL and endothelial lipase. In this process, the larger HDLs are converted to smaller subspecies, which can break apart releasing apoA-I, leading to its renal catabolism by the megalin-cubilin complex. Smaller HDLs can then re-enter the lipidation cycle. Although LDL is the major ultimate product from the lipolysis of VLDL, surface materials from TG-rich particles are transferred to the small circulating HDL ₃ and subsequently esterified by LCAT to generate the larger CE–rich HDL ₂ . HDL ₂ contains twice as many cholesterol molecules per unit of apolipoproteins as does HDL ₃ . HDL ₂ can also be converted back to HDL ₃ by HL.

HDL nomenclature has been continually evolving and can be quite confusing. As preβ-HDL species mature, they may evolve into what a recent expert committee has labeled as very small, small, medium, large, and very large HDL particles. Historically, the small particles have been called HDL ₃ (subtypes a, b, and c, with a being the largest), and the large particles have been called HDL ₂ (subtypes a and b, with b being larger). NMR spectroscopic separation also refers to small, medium, and large particles called H1 to H5, with H5 being the largest. Two-dimensional electrophoretic separation with apoA-I staining classifies HDLs into preβ and α species, with α-4 being the smallest and α-1 being the largest.

Previous research on HDL focused on a possible role in atherosclerosis and ASCVD prevention through RCT; however, genetic studies and failures with therapies aimed at increasing HDL-C now question the role and function of HDL in human health and disease. HDL is the most abundant lipoprotein in plasma of most species, pointing to an important role of HDL in humans. Recent observational studies have shown that extreme elevations of HDL-C are associated with increased mortality, leading to speculations that HDL could in some instances be harmful. In addition, evidence from observational and some genetic studies suggest that HDL concentration might be associated with the development of other major non-cardiovascular diseases such as infectious disease, autoimmune disease, and cancer.

Because of the CE for TG heterotypic exchange between HDL and TG-rich lipoproteins, low HDL-C is a stable marker of average high TG and remnant cholesterol. This suggests that low HDL-C can be used to monitor long-term average high TG and remnant cholesterol, analogous to high HbA1c as a long-term monitor of average high glucose levels.

Primary versus secondary dyslipoproteinemias

When dyslipidemia is first identified, it should be determined whether it is a primary or secondary lipoprotein disorder. Secondary influences include diet, alcohol intake and lifestyle, other diseases, as well as numerous pharmacologic agents, such as steroids, isotretinoin, β-blockers, and antiretroviral agents. The diagnosis of a primary disorder of lipoprotein metabolism is made after secondary causes have been ruled out ( Table 36.18 ), or when the underlying genetic etiology is identified.

Polygenic and monogenic lipoprotein disorders

Most lipoprotein disorders have polygenic or multifactorial causes with small culmulative contributions by many genes or susceptible genes which might have been influenced by environmental factors and familial factors. Through genome wide association studies, a great number of genes have been identified that are associated with various lipid abnormalities.

Monogenic disorders are typically rare, and caused predominantly by variations in a single gene with a large effect. They often have a recognizable familial inheritance pattern. To diagnose monogenic disorders, family history is important, not only for diagnosis, but also for family screening to clearly identify family members at risk. Recent advances in genetic technologies, with expanding availability and decreasing costs, have enabled and increased the ability to identify disease-causing mutations. Therefore it has become feasible to categorize these disorders according to their underlying genetic etiologies.