Cholesterol Absorption

Cholesterol enters the intestinal lumen from three sources: the diet, bile, and the intestine. Animal products—especially meat, egg yolk, seafood, and whole fat dairy products—provide the bulk of dietary cholesterol. Although cholesterol intake varies considerably based on the dietary intake of animal products, the average American diet is estimated to contain approximately 300 to 450 mg of cholesterol per day. A similar amount of cholesterol enters the gut from biliary secretion and from the turnover of intestinal mucosal cells, and possibly from direct intestinal secretions. Practically all cholesterol in the intestinal lumen is present in the unesterified or free form. Esterified cholesterol in the diet is rapidly hydrolyzed in the intestine to free cholesterol and free fatty acids by cholesterol esterases secreted from the pancreas and small intestine.

To be absorbed, unesterified cholesterol first must be solubilized. This occurs through the formation of mixed micelles that contain unesterified cholesterol, fatty acids, monoglycerides (derived from triglycerides), phospholipids, and conjugated bile acids. Formation of mixed micelles promotes cholesterol absorption by both solubilizing the cholesterol and facilitating its transport to the surface of the luminal cell, where it is absorbed by an active process involving the enterocyte protein NPC1L1, which is the drug target for the cholesterol absorption inhibitor ezetimibe. Because of their amphipathic properties, the bile acids act as detergents and are the most important factor affecting micelle formation. In the absence of bile acids, digestion and absorption of both cholesterol and triglyceride are severely impaired, leading to fat malabsorption. The quantity of dietary cholesterol that can be absorbed appears to depend on the amount that can be solubilized by micelles. On average, 30 to 60% of dietary and intestinal cholesterol is absorbed daily. With increments in dietary cholesterol, additional cholesterol is absorbed to a maximum of approximately 1 g/d when oral intake reaches 3 g/d. Absorption of cholesterol and other sterols, such as plant sterols, is limited by the presence of the ABCG5/G8 transporter on enterocytes, which pumps excess sterols back into the lumen for excretion. When this protein is defective, it results in a disease called sitosterolemia, which is characterized by a marked increase in plasma and tissue concentrations of plant sterols, such as sitosterol, and an increased risk for CHD. The ability of cholesterol to form micelles is also influenced by the quantity of dietary fat but not 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. Most cholesterol absorption occurs from the jejunum to the terminal ileum of the small intestine. As absorption of fat and cholesterol occurs in the small intestine, the micelles break up, thus reducing further cholesterol absorption.

After its absorption into the intestinal mucosal cell, cholesterol, together with triglycerides, phospholipids, and a number of specific apoproteins, is assembled into a large lipoprotein called a chylomicron (see later section on lipoprotein metabolism, exogenous pathway). One apoprotein component known as apolipoprotein (apo) B-48 is vital in the formation and secretion of chylomicrons. Patients with a rare deficiency in this process develop a disease called chylomicron retention disorder, which is characterized by excess lipid in enterocytes and fat malabsorption. Once secreted by enterocytes, chylomicrons enter the lymphatics, which eventually empty into the thoracic duct and enter the systemic venous circulation at the junction of the left subclavian vein and the left internal jugular vein.

Cholesterol Synthesis

Although cholesterol enters the body from the diet, it is also synthesized endogenously by all tissues from acetate. Knowledge of the endogenous cholesterol synthetic pathway has assumed great significance, because drug agents for the treatment of CHD have been sought to suppress or decrease endogenous cholesterol synthesis. 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 does not inhibit the rate-limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (Figure 27-2). When triparanol was used to treat hypercholesterolemia, the drug caused tissue accumulation of desmosterol, resulting in the development of cataracts, alopecia, and accelerated atherosclerosis. Many drugs now used to treat CHD selectively suppress the rate-limiting enzyme HMG-CoA reductase, thereby lowering serum cholesterol concentrations significantly without accumulation of water-insoluble intermediates of cholesterol synthesis, such as desmosterol.

Although essentially all cells have the capacity to synthesize cholesterol from acetyl-CoA, almost 90% of synthesis occurs in the liver and gut; other organs and tissues depend, in part, on cholesterol delivery from the circulation. Cholesterol biosynthesis is best conceptualized as occurring in three stages (Figures 27-2 through 27-4). 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 to mevalonate, then is decarboxylated to form five-carbon isoprene units. These isoprene units are condensed to form first a 10-carbon (geranyl pyrophosphate) and then a 15-carbon intermediate (farnesyl pyrophosphate). Two of these C₁₅ molecules combine to produce the final product of the second stage—squalene, a 30-carbon acyclic hydrocarbon. The second stage is important, because it contains the step involving the microsomal enzyme HMG-CoA reductase, which is the rate-limiting enzyme in cholesterol biosynthesis. The enzyme that forms farnesyl pyrophosphate, geranyl transferase, is an important second site of regulation in cholesterol synthesis. This degree of inhibition still permits the formation of physiologically important intermediate isoprenoids in the absence of cholesterol synthesis. The third and final stage of synthesis occurs in the endoplasmic reticulum, with many of the intermediate products being bound to a specific carrier protein. Squalene, after initial oxidation, undergoes cyclization to form the four-ring, 30-carbon intermediate, lanosterol. In a series of oxidation-decarboxylation reactions, several sidechains are removed from the pentanophenanthrene structure to form the final 27-carbon molecule of cholesterol.

Cholesterol Esterification

Once synthesized, cholesterol is released into the circulation bound to lipoproteins, such as very low-density lipoprotein (VLDL; see later section on lipoprotein metabolism, endogenous pathway), which is one of the primary lipoproteins secreted by the liver. The major apoprotein found in VLDL is apo B-100. It is produced by the same gene as apo B-48, but represents the full-length product of the apo B gene. Apo B-48 is produced in the intestine by a post-transcriptional editing step, which introduces a stop codon in about the middle of the apo B-100 mRNA transcript, thus resulting in a protein about 48% the length of the full-length apo B-100 protein. The esterification of cholesterol is important, because it serves to enhance the lipid-carrying capacity of the lipoprotein in plasma and prevents intracellular toxicity by free cholesterol. The reaction is catalyzed by lecithin-cholesterol acyltransferase (LCAT) in the plasma and acylcholesterol acyltransferase (ACAT) within the cell. ACAT is an energy-requiring enzyme, and the initial reaction (Figure 27-5) involves activation of a fatty acid with thio coenzyme A (Co-ASH) to form an acyl-CoA, which in turn reacts with cholesterol to form an ester. The LCAT reaction does not require CoASH and results from fatty acid transfer from the second carbon position of phosphatidyl choline (lecithin) to the hydroxyl group on the A-ring of cholesterol (see Figure 27-5). Cholesteryl esters account for about 70% of the total cholesterol in plasma, and LCAT is responsible for the formation of virtually all plasma cholesteryl ester. LCAT is synthesized in the liver and released into the circulation; it primarily resides on lipoproteins and is activated by apo A-I and other apolipoproteins (see later section on apolipoproteins). The esterification of cholesterol by LCAT on the only polar group of cholesterol makes cholesteryl ester more hydrophobic than cholesterol. This causes the cholesterol on the surface of lipoproteins, once esterified, to partition into the more hydrophobic core of lipoproteins, where triglycerides are also located.

Cholesterol Catabolism

Once a lipoprotein enters the cell, its cholesteryl esters and triglycerides are hydrolyzed by lysosomal acid lipase. Lack or malfunction of this enzyme results in intracellular accumulation of cholesteryl esters and triglycerides, particularly in the liver, and produces a clinical disorder known as cholesteryl ester storage disease, or Wolman’s disease.

Cholesterol reaching the liver may be secreted unchanged into bile or metabolized to bile acids or resecreted on lipoproteins. Approximately one third of the daily production of cholesterol, or about 400 mg/d, is converted into bile acids (Figure 27-6). 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 of the sterol nucleus, catalyzed by the enzyme 7α-hydroxylase (see Figure 27-6). The bile acids are made more polar after conjugation with glycine or taurine and then are excreted into the bile canaliculi. After reaching the small intestine, the conjugated bile acids play an active part in cholesterol and fat absorption, as discussed previously. Some of the bile acids are deconjugated and converted by bacteria in the intestine to secondary bile acids. Cholic acid is converted to deoxycholic acid, and chenodeoxycholic acid is metabolized to lithocholic acid. About 90% of the bile acids, except lithocholic, are reabsorbed in the lower third of the ileum and returned to the liver by the portal vein, which is called the enterohepatic circulation (see also Chapter 50).

A significant amount of cholesterol is also excreted directly into the biliary system, where it is solubilized to form mixed micelles with bile acids and phospholipids. 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, such as the adrenal gland, most cells cannot further catabolize or modify cholesterol. Because of this and its limited aqueous solubility, cholesterol tends to accumulate in cells or extracellular spaces; this is another feature of cholesterol that contributes to its ability to cause atherosclerosis.

Fatty Acid Catabolism

Long-chain fatty acids are oxidized in the mitochondria and produce energy by a series of reactions that operate in a repetitive manner to shorten the fatty acid chain by two carbon atoms at a time from the carboxy (—COOH) terminal of the molecule through a process known as β-oxidation. For example, 1 mole of C₁₆ fatty acid is converted to 8 moles of acetyl-CoA. Acetyl-CoA does not normally accumulate in the cell but is enzymatically condensed with oxaloacetate, derived largely from carbohydrate metabolism (Figure 27-8), to yield citrate, which is a major component of the tricarboxylic acid cycle, also called 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 to carbon dioxide and water produces 16 moles of CO₂, 16 moles of H₂O, and 129 moles of adenosine triphosphate, or 2340 Cal.* 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 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. Triglycerides that contain three fatty acid molecules, therefore, 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 an equivalent amount (2.5 mole) of glucose (6 carbon atoms per molecule). Carbohydrate storage also requires water for hydration; triglyceride storage does not. In addition to their high intrinsic energy content, triglycerides have a low density (<1 g/mL) and, because of their hydrophobic nature and peripheral distribution in the body, provide excellent insulation.

Ketone Formation

During prolonged starvation, or when carbohydrate metabolism is severely impaired, as in uncontrolled diabetes mellitus (see Chapter 26), 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 excessive degradation of fatty acids by β-oxidation in the liver. The resulting acetyl-CoA excess is diverted to an alternative pathway in the mitochondria and forms acetoacetic acid, β-hydroxybutyric acid, and acetone—three compounds known collectively as ketone bodies (Figure 27-9). The presence of ketone bodies is a frequent finding in severe, uncontrolled diabetes mellitus.

As shown in Figure 27-9, 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 usually reduced to β-hydroxybutyrate. Because acetoacetate is unstable, a further portion decomposes to form carbon dioxide and acetone, the third ketone body found in severe, untreated diabetes mellitus. Ketosis, therefore, develops from excessive production of acetyl-CoA because the body attempts to derive necessary energy from stored fat in the absence of an adequate supply of carbohydrate metabolites (see Chapter 26).

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 heart (and 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 converting fatty acids, 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 ketosis is reversed by restoring adequate metabolism of carbohydrate. In starvation, restoration consists of adequate carbohydrate ingestion; in diabetes mellitus, 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 instead 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 Figure 27-8, which shows the interrelationship between carbohydrate, fatty acid, and protein metabolism.

Prostaglandins

Prostaglandins and related compounds are derivatives of fatty acids, primarily arachidonate. This group consists of prostaglandins, thromboxanes, some hydroperoxy– and hydroxy–fatty acid derivatives, and leukotrienes. Although their full physiologic role is not completely known, these lipids are known to affect a wide variety of biological functions. In general, they are extremely potent, producing physiologic actions at concentrations as low as 1 µg/L.

The prostaglandins are a series of C₂₀ unsaturated fatty acids containing a cyclopentane ring; the parent fatty acid has been given the trivial name prostanoic acid. The seven-carbon chain linked to C-8 of prostanoic acid (R₁) projects below the plane of the ring, whereas the eight-carbon chain attached to C-12 (R₂) projects above the ring.

By convention, prostaglandins are abbreviated PG, with the class designated by a capital letter (A, B, E, F, G, H, and I), followed by a number and then in some cases a Greek letter (Figure 27-10). With the exception of PGG and PGH, which have the same ring structure (cyclopentane endoperoxide), the letters refer to different ring structures. PGA and PGB have keto groups at C-9, with the A series having a double bond between C-10 and C-11, and the B series having a double bond between C-8 and C-12. PGE also has a C-9 keto bond but a hydroxyl group at C-11. The F series has hydroxyl groups at both C-9 and C-11. The difference between PGG and PGH, which have identical ring structures, occurs in the sidechain at C-15 in R₂. The G series has a peroxide group, whereas the H series has a hydroxyl group. The I series has a double-ring formation, with the C-9 of the cyclopentane ring linked to C-6 of the sidechain by an oxygen molecule to form a second five-sided ring (see Figure 27-10). The endoperoxide PGs (G and H series) are intermediates in the formation of other PGs.

The number after the capital letter is usually written as a subscript and is used to designate the number of unsaturated bonds in the PG sidechains and not within the ring structure itself. In PGE₁, for example, a double bond exists between C-13 and C-14; in the 2 series (PGE₂), a double bond exists between C-13 and C-14 and between C-5 and C-6; and in the 3 series (PGE₃), an additional double bond occurs between C-17 and C-18. The 2 series is most common. The bond between C-13 and C-14 is always trans, whereas bonds between C-5 and C-6 and between C-17 and C-18 are always cis. At C-15, all naturally occurring prostaglandins have a hydroxyl group that projects below the plane of the ring. Use of the Greek letter (α or β) applies only to the F series and refers to the hydroxyl group found at C-9. In the α-series, the hydroxyl group projects below the ring plane in the same direction as the C-11 hydroxyl group, whereas the β-series denotes that the hydroxyl at C-9 is above the plane of the ring. Sixteen naturally occurring prostaglandins have been described (Table 27-2), but only seven, along with two thromboxanes, are commonly found throughout the body, and they are termed the primary prostaglandins.

Although prostaglandins have hormone-like actions, they are different from most other hormones in that (1) they are synthesized at the site of action, and (2) they are made in almost all tissues. Linoleic acid (C₁₈:29,12) is the precursor of two of the three 20-carbon fatty acids that form prostaglandins; linolenic acid (C₁₈:2^9,12,15) is the other precursor. Both of these fatty acids are considered essential because they cannot be synthesized in the body and, therefore, must be present in the diet. The three C₂₀ fatty acids subsequently formed are C₂₀:3^5,8,11 (eicosatrienoic acid), C₂₀:4^5,8,11,14 (eicosatetraenoic or arachidonic acid), and C₂₀:5^8,11,14,17(eicosatrienoic acid). These three fatty acids form the PG₁, PG₂, and PG₃ series, respectively.

Once formed, prostaglandins exert very short-lived effects and are rapidly catabolized to inactive forms within a few seconds. Inactivation of prostaglandin appears to be mediated by two enzymes: 15 α-hydroxy-prostaglandin dehydrogenase and Δ¹³-prostaglandin reductase. Prostaglandins are not stored; instead, precursor C₂₀ fatty acids are present in tissue attached to the C-2 (see later section in this chapter on glycerol esters) of phosphoglycerides. When necessary, the C₂₀ precursor is hydrolyzed by phospholipase A₂, which is specific for the C-2 position of phosphoglyceride. Release of the C₂₀ fatty acid appears to be the rate-limiting step in prostaglandin synthesis and is stimulated by many different agents, such as bradykinin, thrombin, or angiotensin II.

Although it is probable that all prostaglandins follow a similar synthetic pathway, C₂₀:4 (arachidonic acid) has been the most intensively studied and is used to illustrate the pathway (Figure 27-11). Once released, arachidonic acid follows one of two pathways. The lipoxygenase route produces 12-L-hydroperoxy-5,8,10,14 eicosatetraenoic acid (HPETE); HPETE then spontaneously decomposes to 12-L-hydroxy-5,8,10,14 eicosatetraenoic acid (HETE). The alternative pathway is mediated by cyclooxygenase (COX) to produce the endoperoxides PGG₂ and PGH₂. The latter can be degraded to 12-L-hydroxy-5,8,10-heptadecatrienoic acid. What controls the entry into a specific pathway remains speculative; however, it is known that nonsteroidal anti-inflammatory drugs (NSAIDs; aspirin, ibuprofen, and indomethacin) inhibit the COXs, thereby decreasing prostaglandin synthesis. Two isoforms of COX are known: COX-1 and COX-2. COX-1 concentrations in general are constitutively expressed, whereas COX-2 is synthesized in response to inflammation. Certain drugs that inhibit both COXs have nephrotoxic and ulcerogenic side effects. Therefore, newer NSAIDs have been developed that preferentially inhibit COX-2 to reduce side effects, but they have been found to also be associated with an increased incidence of myocardial infarction.⁹⁴

Prostaglandin I₂, or prostacyclin, is derived from arachidonic acid (see Figure 27-11) in the vascular endothelium. It has a powerful vasodilatory action, especially on the coronary arteries, and is responsible for inhibiting platelet aggregation. Thromboxane A₂ is synthesized from arachidonic acid but is also produced by platelets. It has the opposite effect of prostacyclin (i.e., it stimulates the contraction of arterial smooth muscle and enhances platelet aggregation). It has a very short half-life—about 30 seconds—and is rapidly converted to its inactive metabolite, thromboxane B₂. The thromboxanes are slightly different from other prostaglandins in that they contain six-sided rings of five carbon atoms and one oxygen atom (Figure 27-12). Table 27-3 lists some reported functions of the various prostaglandins.