Biochemistry_Lippinco

Biochemistry_Lippinco. III. FATTY ACID DE NOVO SYNTHESIS Carbohydrates and proteins obtained from the diet in excess of the body’s needs for these nutrients can be converted to fatty acids. In adults, de novo fatty acid synthesis occurs primarily in the liver and lactating mammary glands and, to a lesser extent, in adipose tissue. This cytosolic process is endergonic (see p. 70) and reductive. It incorporates carbons from acetyl coenzyme A (CoA) into the growing fatty acid chain, using ATP and reduced nicotinamide adenine dinucleotide phosphate (NADPH). [Note: Dietary TAG also supply fatty acids. See p. 321 for a discussion of the metabolism of dietary nutrients in the well-fed state.] A. Cytosolic acetyl CoA production


Biochemistry_Lippinco

Biochemistry_Lippinco. The first step in fatty acid synthesis is the transfer of acetate units from mitochondrial acetyl CoA to the cytosol. Mitochondrial acetyl CoA is produced by the oxidation of pyruvate (see p. 109) and by the catabolism of certain amino acids (see p. 266). However, the CoA portion of acetyl CoA cannot cross the inner mitochondrial membrane, and only the acetyl portion enters the cytosol. It does so as part of citrate produced by the condensation of acetyl CoA with oxaloacetate (OAA) by citrate synthase (Fig. 16.6). [Note: The transport of citrate to the cytosol occurs when the mitochondrial citrate concentration is high. This is observed when isocitrate dehydrogenase of the tricarboxylic acid (TCA) cycle is inhibited by the presence of large amounts of ATP, causing citrate and isocitrate to accumulate (see p. 112). Therefore, cytosolic citrate may be viewed as a high-energy signal. Because a large amount of ATP is needed for fatty acid synthesis, the increase in both ATP and citrate


Biochemistry_Lippinco

Biochemistry_Lippinco. to accumulate (see p. 112). Therefore, cytosolic citrate may be viewed as a high-energy signal. Because a large amount of ATP is needed for fatty acid synthesis, the increase in both ATP and citrate enhances this pathway.] In the cytosol, citrate is cleaved to OAA and acetyl CoA by ATP citrate lyase.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Acetyl CoA carboxylation to malonyl CoA The energy for the carbon-to-carbon condensations in fatty acid synthesis is supplied by the carboxylation and then decarboxylation of acyl groups in the cytosol. The carboxylation of acetyl CoA to malonyl CoA is catalyzed by acetyl CoA carboxylase (ACC ) (Fig. 16.7). ACC transfers carbon dioxide (CO2) from bicarbonate ( ) in an ATP-requiring reaction. The coenzyme is biotin (vitamin B7), which is covalently bound to a lysyl residue of the carboxylase (see Fig. 28.16, p. 385). ACC carboxylates the bound biotin, which transfers the activated carboxyl group to acetyl CoA. acetylCoAcarboxylase. diphosphate.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Acetyl CoA carboxylase short-term regulation: This carboxylation is both the rate-limiting and the regulated step in fatty acid synthesis (see Fig. 16.7). The inactive form of ACC is a protomer (complex of ≥2 polypeptides). The enzyme is allosterically activated by citrate, which causes protomers to polymerize, and allosterically inactivated by palmitoyl CoA (the end product of the pathway), which causes depolymerization. A second mechanism of short-term regulation is by reversible phosphorylation. Adenosine monophosphate–activated protein kinase (AMPK) phosphorylates and inactivates ACC. AMPK itself is activated allosterically by AMP and covalently by phosphorylation via several kinases. At least one of these AMPK kinases is activated by cyclic AMP (cAMP)–dependent protein kinase A (PKA). Thus, in the presence of counterregulatory hormones, such as epinephrine and glucagon, ACC is phosphorylated and inactive (Fig. 16.8). In the presence of insulin, ACC is dephosphorylated and


Biochemistry_Lippinco

Biochemistry_Lippinco. A (PKA). Thus, in the presence of counterregulatory hormones, such as epinephrine and glucagon, ACC is phosphorylated and inactive (Fig. 16.8). In the presence of insulin, ACC is dephosphorylated and active. [Note: This is analogous to the regulation of glycogen synthase (see p. 131).] itself is regulated both covalently and allosterically. CoA = coenzyme A; ADP phosphate.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Acetyl CoA carboxylase long-term regulation: Prolonged consumption of a diet containing excess calories (particularly high-carbohydrate, low-fat diets) causes an increase in ACC synthesis, thereby increasing fatty acid synthesis. A low-calorie or a high-fat, low-carbohydrate diet has the opposite effect. [Note: ACC synthesis is upregulated by carbohydrate (specifically glucose) via the transcription factor carbohydrate response element–binding protein (ChREBP) and by insulin via the transcription factor sterol regulatory element–binding protein-1c (SREBP-1c). Fatty acid synthase (see C. below) is similarly regulated. The function and regulation of SREBP are described on p. 222.] Metformin, used in the treatment of type 2 diabetes, lowers plasma TAG through activation of AMPK, resulting in inhibition of ACC activity (by phosphorylation) and inhibition of ACC and fatty acid synthase expression (by decreasing SREBP-1c). Metformin lowers blood glucose by increasing AMPK-mediated


Biochemistry_Lippinco

Biochemistry_Lippinco. in inhibition of ACC activity (by phosphorylation) and inhibition of ACC and fatty acid synthase expression (by decreasing SREBP-1c). Metformin lowers blood glucose by increasing AMPK-mediated glucose uptake by muscle.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Eukaryotic fatty acid synthase The remaining series of reactions of fatty acid synthesis in eukaryotes is catalyzed by the multifunctional, homodimeric enzyme fatty acid synthase (FAS). The process involves the addition of two carbons from malonyl CoA to the carboxyl end of a series of acyl acceptors. Each FAS monomer is a multicatalytic polypeptide with six different enzymic domains plus a 4ʹphosphopantetheine-containing acyl carrier protein (ACP) domain. 4ʹ-Phosphopantetheine, a derivative of pantothenic acid (vitamin B5, see p. 385), carries acyl units on its terminal thiol (–SH) group and presents them to the catalytic domains of FAS during fatty acid synthesis. It also is a component of CoA. [Note: In prokaryotes, FAS is a multienzyme complex.] The reaction numbers in brackets below refer to Figure 16.9. adenine dinucleotide phosphate. 1. An acetyl group is transferred from acetyl CoA to the –SH group of the ACP. Domain: Malonyl/acetyl CoA–ACP transacylase. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. adenine dinucleotide phosphate. 1. An acetyl group is transferred from acetyl CoA to the –SH group of the ACP. Domain: Malonyl/acetyl CoA–ACP transacylase. 2. Next, this two-carbon fragment is transferred to a temporary holding site, the –SH group of a cysteine residue on the condensing enzyme domain (see [4] below). 3. The now-vacant ACP accepts a three-carbon malonyl group from malonyl CoA. Domain: Malonyl/acetyl CoA–ACP transacylase. 4. The acetyl group on the cysteine residue condenses with the malonyl group on ACP as the CO2 originally added by ACC is released. The result is a four-carbon unit attached to the ACP domain. The loss of free energy from the decarboxylation drives the reaction. Domain: 3Ketoacyl–ACP synthase, also known as condensing enzyme. The next three reactions convert the 3-ketoacyl group to the corresponding saturated acyl group by a pair of NADPH-requiring reductions and a dehydration step. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. The next three reactions convert the 3-ketoacyl group to the corresponding saturated acyl group by a pair of NADPH-requiring reductions and a dehydration step. 1. The keto group is reduced to an alcohol. Domain: 3-Ketoacyl–ACP reductase. 2. A molecule of water is removed, creating a trans double bond between carbons 2 and 3 (the α-and β-carbons). Domain: 3-Hydroxyacyl–ACP dehydratase. 3. The double bond is reduced. Domain: Enoyl–ACP reductase.


Biochemistry_Lippinco

Biochemistry_Lippinco. This sequence of steps results in the production of a four-carbon group (butyryl) whose three terminal carbons are fully saturated and which remains attached to the ACP domain. The steps are repeated (indicated by an asterisk), beginning with the transfer of the butyryl unit from the ACP to the cysteine residue [2*], the attachment of a malonyl group to the ACP [3*], and the condensation of the two groups liberating CO2 [4*]. The carbonyl group at the β-carbon (carbon 3, the third carbon from the sulfur) is then reduced [5*], dehydrated [6*], and reduced [7*], generating hexanoyl-ACP. This cycle of reactions is repeated five more times, each time incorporating a two-carbon unit (derived from malonyl CoA) into the growing fatty acid chain at the carboxyl end. When the fatty acid reaches a length of 16 carbons, the synthetic process is terminated with palmitoyl-S-ACP. [Note: Shorter-length fatty acids are produced in the lactating mammary gland.] Palmitoyl thioesterase, the final


Biochemistry_Lippinco

Biochemistry_Lippinco. a length of 16 carbons, the synthetic process is terminated with palmitoyl-S-ACP. [Note: Shorter-length fatty acids are produced in the lactating mammary gland.] Palmitoyl thioesterase, the final catalytic activity of FAS, cleaves the thioester bond, releasing a fully saturated molecule of palmitate (16:0). [Note: All the carbons in palmitic acid have passed through malonyl CoA except the two donated by the original acetyl CoA (the first acyl acceptor), which are found at the methyl (ω) end of the fatty acid. This underscores the rate-limiting nature of the ACC reaction.]


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Reductant sources The synthesis of one palmitate requires 14 NADPH, a reductant (reducing agent). The pentose phosphate pathway (see p. 145) is a major supplier of the NADPH. Two NADPH are produced for each molecule of glucose 6phosphate that enters this pathway. The cytosolic conversion of malate to pyruvate, in which malate is oxidized and decarboxylated by cytosolic malic enzyme (NADP+-dependent malate dehydrogenase), also produces cytosolic NADPH (and CO2), as shown in Figure 16.10. [Note: Malate can arise from the reduction of OAA by cytosolic NADH-dependent malate dehydrogenase (see Fig. 16.10). One source of the cytosolic NADH required for this reaction is glycolysis (see p. 101). OAA, in turn, can arise from citrate cleavage by ATP citrate lyase.] A summary of the interrelationship between glucose metabolism and palmitate synthesis is shown in Figure 16.11. tricarboxylic acid; PC = pyruvate carboxylase; PDH = pyruvate dehydrogenase. E. Further elongation


Biochemistry_Lippinco

Biochemistry_Lippinco. tricarboxylic acid; PC = pyruvate carboxylase; PDH = pyruvate dehydrogenase. E. Further elongation Although palmitate, a 16-carbon, fully saturated LCFA (16:0), is the primary end product of FAS activity, it can be further elongated by the addition of two-carbon units to the carboxylate end primarily in the smooth endoplasmic reticulum (SER). Elongation requires a system of separate enzymes rather than a multifunctional enzyme. Malonyl CoA is the two-carbon donor, and NADPH supplies the electrons. The brain has additional elongation capabilities, allowing it to produce the very-long-chain fatty acids ([VLCFA] over 22 carbons) that are required for synthesis of brain lipids. F. Chain desaturation Enzymes (fatty acyl CoA desaturases) also present in the SER are responsible for desaturating LCFA (that is, adding cis double bonds). The desaturation reactions require oxygen (O2), NADH, cytochrome b5, and its flavin adenine dinucleotide (FAD)-linked reductase. The fatty acid and the


Biochemistry_Lippinco

Biochemistry_Lippinco. NADH get oxidized as the O2 gets reduced to H2O. The first double bond is typically inserted between carbons 9 and 10, producing primarily oleic acid, 18:1(9), and small amounts of palmitoleic acid, 16:1(9). A variety of polyunsaturated fatty acids can be made through additional desaturation combined with elongation. Humans have carbon 9, 6, 5, and 4 desaturases but lack the ability to introduce double bonds from carbon 10 to the ω end of the chain. This is the basis for the nutritional essentiality of the polyunsaturated ω-6 linoleic acid and ω-3 linolenic acid. G. Storage as triacylglycerol components


Biochemistry_Lippinco

Biochemistry_Lippinco. G. Storage as triacylglycerol components Mono-, di-, and triacylglycerols consist of one, two, or three molecules of fatty acid esterified to a molecule of glycerol. Fatty acids are esterified through their carboxyl groups, resulting in a loss of negative charge and formation of neutral fat. [Note: An acylglycerol that is solid at room temperature is called a fat. If liquid, it is an oil.] 1. Arrangement: The three fatty acids esterified to a glycerol molecule to form a TAG are usually not of the same type. The fatty acid on carbon 1 is typically saturated, that on carbon 2 is typically unsaturated, and that on carbon 3 can be either. Recall that the presence of the unsaturated fatty acid(s) decrease(s) the Tm of the lipid. An example of a TAG molecule is shown in Figure 16.12. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Triacylglycerol storage and function: Because TAG are only slightly soluble in water and cannot form stable micelles by themselves, they coalesce within white adipocytes to form large oily droplets that are nearly anhydrous. These cytosolic lipid droplets are the major energy reserve of the body. [Note: TAG stored in brown adipocytes serve as a source of heat through nonshivering thermogenesis (see p. 79).] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Glycerol 3-phosphate synthesis: Glycerol 3-phosphate is the initial acceptor of fatty acids during TAG synthesis. There are two major pathways for its production (Fig. 16.13). [Note: A third process (glyceroneogenesis) is described on p. 190.] In both liver (the primary site of TAG synthesis) and adipose tissue, glycerol 3-phosphate can be produced from glucose, first using the reactions of the glycolytic pathway to produce dihydroxyacetone phosphate ([DHAP], see p. 101). DHAP is reduced by glycerol 3-phosphate dehydrogenase to glycerol 3phosphate. A second pathway found in the liver, but not in adipose tissue, uses glycerol kinase to convert free glycerol to glycerol 3phosphate (see Fig. 16.13). [Note: The glucose transporter in adipocytes (GLUT-4) is insulin dependent (see p. 312). Thus, when plasma glucose levels are low, adipocytes have only a limited ability to synthesize glycerol phosphate and cannot produce TAG de novo.] 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. Fatty acid activation: A free fatty acid must be converted to its activated form (bound to CoA through a thioester link) before it can participate in metabolic processes such as TAG synthesis. This reaction, illustrated in Figure 15.6 on p. 177, is catalyzed by a family of fatty acyl CoA synthetases (thiokinases). 5. Triacylglycerol synthesis: This pathway from glycerol 3-phosphate involves four reactions, shown in Figure 16.14. These include the sequential addition of two fatty acids from fatty acyl CoA, the removal of phosphate, and the addition of the third fatty acid. H. Triacylglycerol fate in liver and adipose tissue


Biochemistry_Lippinco

Biochemistry_Lippinco. H. Triacylglycerol fate in liver and adipose tissue In WAT, TAG is stored in a nearly anhydrous form as fat droplets in the cytosol of the cells. It serves as “depot fat,” ready for mobilization when the body requires it for fuel. Little TAG is stored in healthy liver. Instead, most is exported, packaged with other lipids and apolipoproteins to form lipoprotein particles called very-low-density lipoproteins (VLDL). Nascent VLDL are secreted directly into the blood where they mature and function to deliver the endogenously derived lipids to the peripheral tissues. [Note: Recall from Chapter 15 that chylomicrons carry dietary (exogenously derived) lipids. Plasma lipoproteins are discussed in Chapter 18.] IV. FAT MOBILIZATION AND FATTY ACID OXIDATION


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. FAT MOBILIZATION AND FATTY ACID OXIDATION Fatty acids stored in WAT, in the form of neutral TAG, serve as the body’s major fuel storage reserve. TAG provide concentrated stores of metabolic energy because they are highly reduced and largely anhydrous. The yield from the complete oxidation of fatty acids to CO2 and H2O is 9 kcal/g fat (as compared to 4 kcal/g protein or carbohydrate, see Fig. 27.5 on p. 359). A. Fatty acid release from fat The mobilization of stored fat requires the hydrolytic release of FFA and glycerol from their TAG form. This process of lipolysis is achieved by lipases. It is initiated by adipose triglyceride lipase (ATGL), which generates a diacylglycerol that is the preferred substrate for hormone-sensitive lipase (HSL). The monoacylglycerol (MAG) product of HSL is acted upon by MAG lipase.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Hormone-sensitive lipase regulation: HSL is active when phosphorylated by PKA, a cAMP-dependent protein kinase. cAMP is produced in the adipocyte when catecholamines (such as epinephrine) bind to cell membrane β-adrenergic receptors and activate adenylyl cyclase (Fig. 16.15). The process is similar to that of the activation of glycogen phosphorylase (see Fig. 11.9, p. 131). [Note: Because ACC is inhibited by hormone-directed phosphorylation, when the cAMP-mediated cascade is activated (see Fig. 16.8), fatty acid synthesis is turned off and TAG degradation is turned on.] In the presence of high plasma levels of insulin, HSL is dephosphorylated and inactivated. Insulin also suppresses expression of ATGL. [Note: Fat droplets are coated by a protein (perilipin) that limits access of HSL. Phosphorylation of perilipin by PKA allows translocation and binding of phosphorylated HSL to the droplet.] pyrophosphate; ADP = adenosine diphosphate; = phosphate. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Fate of glycerol: The glycerol released during TAG degradation cannot be metabolized by adipocytes because they lack glycerol kinase. Rather, glycerol is transported through the blood to the liver, which has the kinase. The resulting glycerol 3-phosphate can be used to form TAG in the liver or can be converted to DHAP by reversal of the glycerol 3phosphate dehydrogenase reaction illustrated in Figure 16.13. DHAP can participate in glycolysis or gluconeogenesis. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Fate of fatty acids: The FFA move through the cell membrane of the adipocyte and bind to serum albumin. They are transported to tissues such as muscle, enter cells, get activated to their CoA derivatives, and are oxidized for energy in mitochondria. Regardless of their levels, plasma FFA cannot be used for fuel by red blood cells (RBC), which have no mitochondria. The brain does not use fatty acids for energy to any appreciable extent, but the reasons are less clear. [Note: Over 50% of the fatty acids released from adipose TAG are reesterified to glycerol 3phosphate. WAT does not express glycerol kinase, and the glycerol 3phosphate is produced by glyceroneogenesis, an incomplete version of gluconeogenesis: pyruvate to OAA via pyruvate carboxylase and OAA to phosphoenolpyruvate (PEP) via phosphoenolpyruvate carboxykinase. The PEP is converted (by reactions common to glycolysis and gluconeogenesis) to DHAP, which is reduced to glycerol 3-phosphate. The process decreases plasma FFA,


Biochemistry_Lippinco

Biochemistry_Lippinco. phosphoenolpyruvate carboxykinase. The PEP is converted (by reactions common to glycolysis and gluconeogenesis) to DHAP, which is reduced to glycerol 3-phosphate. The process decreases plasma FFA, molecules associated with insulin resistance in type 2 diabetes and obesity (see p. 343).]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Fatty acid β-oxidation The major pathway for catabolism of fatty acids is a mitochondrial pathway called β-oxidation, in which two-carbon fragments are successively removed from the carboxyl end of the fatty acyl CoA, producing acetyl CoA, NADH, and FADH2. 1. Long-chain fatty acid transport into mitochondria: After a LCFA enters a cell, it is converted in the cytosol to its CoA derivative by long-chain fatty acyl CoA synthetase (thiokinase), an enzyme of the outer mitochondrial membrane. Because β-oxidation occurs in the mitochondrial matrix, the fatty acid must be transported across the inner mitochondrial membrane that is impermeable to CoA. Therefore, a specialized carrier transports the long-chain acyl group from the cytosol into the mitochondrial matrix. This carrier is carnitine, and this rate-limiting transport process is called the carnitine shuttle (Fig. 16.16). a.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Translocation steps: First, the acyl group is transferred from CoA to carnitine by carnitine palmitoyltransferase I (CPT-I), an enzyme of the outer mitochondrial membrane. [Note: CPT-I is also known as CAT-I for carnitine acyltransferase I.] This reaction forms an acylcarnitine and regenerates free CoA. Second, the acylcarnitine is transported into the mitochondrial matrix in exchange for free carnitine by carnitine–acylcarnitine translocase. Carnitine palmitoyltransferase 2 (CPT-II, or CAT-II), an enzyme of the inner mitochondrial membrane, catalyzes the transfer of the acyl group from carnitine to CoA in the mitochondrial matrix, thus regenerating free carnitine. b.


Biochemistry_Lippinco

Biochemistry_Lippinco. b. Carnitine shuttle inhibitor: Malonyl CoA inhibits CPT-I, thus preventing the entry of long-chain acyl groups into the mitochondrial matrix. Therefore, when fatty acid synthesis is occurring in the cytosol (as indicated by the presence of malonyl CoA), the newly made palmitate cannot be transferred into mitochondria and degraded. [Note: Muscle tissue, although it does not synthesize fatty acids, contains the mitochondrial isozyme of ACC (ACC2), allowing regulation of βoxidation. The liver contains both isozymes.] Fatty acid oxidation is also regulated by the acetyl CoA/CoA ratio: As the ratio increases, the CoA-requiring thiolase reaction decreases (Fig. 16.17). nicotinamide adenine dinucleotide. c.


Biochemistry_Lippinco

Biochemistry_Lippinco. nicotinamide adenine dinucleotide. c. Carnitine sources: Carnitine can be obtained from the diet, where it is found primarily in meat products. It can also be synthesized from the amino acids lysine and methionine by an enzymatic pathway found in the liver and kidneys but not in skeletal or cardiac muscle. Therefore, these latter tissues are totally dependent on uptake of carnitine provided by endogenous synthesis or the diet and distributed by the blood. [Note: Skeletal muscle contains ~97% of all carnitine in the body.] d.


Biochemistry_Lippinco

Biochemistry_Lippinco. Carnitine deficiencies: Such deficiencies result in decreased ability of tissues to use LCFA as a fuel. Primary carnitine deficiency is caused by defects in a membrane transporter that prevent uptake of carnitine by cardiac and skeletal muscle and the kidneys, causing carnitine to be excreted. Treatment includes carnitine supplementation. Secondary carnitine deficiency occurs primarily as a result of defects in fatty acid oxidation leading to the accumulation of acylcarnitines that are excreted in the urine, decreasing carnitine availability. Acquired secondary carnitine deficiency can be seen, for example, in patients with liver disease (decreased carnitine synthesis) or those taking the antiseizure drug valproic acid (decreased renal reabsorption). [Note: Defects in mitochondrial oxidation can also be caused by deficiencies in CPT-I and CPT-II. CPT-I deficiency affects the liver, where an inability to use LCFA for fuel greatly impairs that tissue’s ability to synthesize glucose (an


Biochemistry_Lippinco

Biochemistry_Lippinco. can also be caused by deficiencies in CPT-I and CPT-II. CPT-I deficiency affects the liver, where an inability to use LCFA for fuel greatly impairs that tissue’s ability to synthesize glucose (an endergonic process) during a fast. This can lead to severe hypoglycemia, coma, and death. CPT-II deficiency can affect the liver and cardiac and skeletal muscle. The most common (and least severe) form affects skeletal muscle. It presents as muscle weakness with myoglobinemia following prolonged exercise. Treatment includes avoidance of fasting and adopting a diet high in carbohydrates and low in fat but supplemented with medium-chain TAG.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Shorter-chain fatty acid entry into mitochondria: Fatty acids ≤12 carbons can cross the inner mitochondrial membrane without the aid of carnitine or the CPT system. Once inside the mitochondria, they are activated to their CoA derivatives by matrix enzymes and are oxidized. [Note: Medium-chain fatty acids are plentiful in human milk. Because their oxidation is not dependent on CPT-I, malonyl CoA is not inhibitory.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. β-Oxidation reactions: The first cycle of β-oxidation is shown in Figure 16.17. It consists of a sequence of four reactions involving the β-carbon (carbon 3) that results in shortening the fatty acid by two carbons at the carboxylate end. The steps include an oxidation that produces FADH2, a hydration, a second oxidation that produces NADH, and a CoAdependent thiolytic cleavage that releases a molecule of acetyl CoA. Each step is catalyzed by enzymes with chain-length specificity. [Note: For LCFA, the last three steps are catalyzed by a trifunctional protein.] These four steps are repeated for saturated fatty acids of even-numbered carbon chains (n/2) − 1 times (where n is the number of carbons), each cycle producing one acetyl CoA plus one NADH and one FADH2. The final cycle produces two acetyl CoA. The acetyl CoA can be oxidized or used in hepatic ketogenesis (see V. below). The reduced coenzymes are oxidized by the electron transport chain, NADH by Complex I, and FADH2 by coenzyme


Biochemistry_Lippinco

Biochemistry_Lippinco. acetyl CoA. The acetyl CoA can be oxidized or used in hepatic ketogenesis (see V. below). The reduced coenzymes are oxidized by the electron transport chain, NADH by Complex I, and FADH2 by coenzyme Q (see p. 75). [Note: Acetyl CoA is a positive allosteric effector of pyruvate carboxylase (see p. 119), thus linking fatty acid oxidation and gluconeogenesis.] 4. β-Oxidation energy yield: The energy yield from fatty acid β-oxidation is high. For example, the oxidation of a molecule of palmitoyl CoA to CO2 and H2O produces 8 acetyl CoA, 7 NADH, and 7 FADH2, from which 131 ATP can be generated. However, activation of the fatty acid requires two ATP. Therefore, the net yield from palmitate is 129 ATP (Fig. 16.18). A comparison of the processes of synthesis and degradation of long-chain saturated fatty acids with an even number of carbon atoms is provided in Figure 16.19.


Biochemistry_Lippinco

Biochemistry_Lippinco. adenine dinucleotide; NADH = nicotinamide adenine dinucleotide; TCA = tricarboxylic acid; CoQ = coenzyme Q; CO2 = carbon dioxide. 5. Medium-chain fatty acyl CoA dehydrogenase deficiency: In mitochondria, there are four fatty acyl CoA dehydrogenase species, each with distinct but overlapping specificity for either short-, medium-, long-, or very-long-chain fatty acids. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency, an autosomal-recessive disorder, is the most common inborn error of β-oxidation, being found in 1:14,000 births worldwide, with a higher incidence in Caucasians of Northern European descent. It results in decreased ability to oxidize fatty acids with six to ten carbons (which accumulate and can be measured in urine), severe hypoglycemia (because the tissues must increase their reliance on glucose), and hypoketonemia (because of decreased production of acetyl CoA; see p. 195). Treatment includes avoidance of fasting. 6.


Biochemistry_Lippinco

Biochemistry_Lippinco. 6. Oxidation of fatty acids with an odd number of carbons: This process proceeds by the same reaction steps as that of fatty acids with an even number of carbons, until the final three carbons are reached. This product, propionyl CoA, is metabolized by a three-step pathway (Fig. 16.20). [Note: Propionyl CoA is also produced during the metabolism of certain amino acids (see Fig. 20.11, p. 266).] a. d-Methylmalonyl CoA synthesis: First, propionyl CoA is carboxylated, forming D-methylmalonyl CoA. The enzyme propionyl CoA carboxylase has an absolute requirement for the coenzymes biotin and ATP, as do ACC and most other carboxylases. b. l-Methylmalonyl CoA formation: Next, the D-isomer is converted to the L-form by the enzyme methylmalonyl CoA racemase. c.


Biochemistry_Lippinco

Biochemistry_Lippinco. c. Succinyl CoA synthesis: Finally, the carbons of L-methylmalonyl CoA are rearranged, forming succinyl CoA, which can enter the TCA cycle (see p. 113). [Note: This is the only example of a glucogenic precursor generated from fatty acid oxidation.] The enzyme methylmalonyl CoA mutase requires a coenzyme form of vitamin B12 (deoxyadenosylcobalamin). The mutase reaction is one of only two reactions in the body that require vitamin B12 (see p. 379). [Note: In patients with vitamin B12 deficiency, both propionate and methylmalonate are excreted in the urine. Two types of heritable methylmalonic acidemia and aciduria have been described: one in which the mutase is missing or deficient (or has reduced affinity for the coenzyme) and one in which the patient is unable to convert vitamin B12 into its coenzyme form. Either type results in metabolic acidosis and neurologic manifestations.] 7.


Biochemistry_Lippinco

Biochemistry_Lippinco. Unsaturated fatty acid β-oxidation: The oxidation of unsaturated fatty acids generates intermediates that cannot serve as substrates for 2,3-enoyl CoA hydratase (see Fig. 16.17). Consequently, additional enzymes are required. Oxidation of a double bond at an odd-numbered carbon, such as 18:1(9) (oleic acid), requires one additional enzyme, 3,2-enoyl CoA isomerase, which converts the 3-cis derivative obtained after three rounds of β-oxidation to the 2-trans derivative required by the hydratase. Oxidation of a double bond at an even-numbered carbon, such as 18:2(9,12) (linoleic acid), requires an NADPH-dependent 2,4-dienoyl CoA reductase in addition to the isomerase. [Note: Because unsaturated fatty acids are less reduced than saturated fatty acids, fewer reducing equivalents are produced by their oxidation.] 8.


Biochemistry_Lippinco

Biochemistry_Lippinco. Peroxisomal β-oxidation: VLCFA ≥22 carbons in length undergo a preliminary β-oxidation in peroxisomes, because peroxisomes and not mitochondria are the primary site of the synthetase that activates fatty acids of this length. The shortened fatty acid (linked to carnitine) diffuses to a mitochondrion for further oxidation. In contrast to mitochondrial βoxidation, the initial dehydrogenation in peroxisomes is catalyzed by a FAD-containing acyl CoA oxidase. The FADH2 produced is oxidized by O2, which is reduced to hydrogen peroxide (H2O2). Therefore, no ATP is generated from this step. The H2O2 is reduced to H2O by catalase (see p. 148). [Note: Genetic defects in the ability either to target matrix proteins to peroxisomes (resulting in Zellweger syndrome, a peroxisomal biogenesis disorder) or to transport VLCFA across the peroxisomal membrane (resulting in X-linked adrenoleukodystrophy) lead to accumulation of VLCFA in the blood and tissues.] C. Peroxisomal α-oxidation


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Peroxisomal α-oxidation Branched-chain phytanic acid, a product of chlorophyll metabolism, is not a substrate for acyl CoA dehydrogenase because of the methyl group on its βcarbon (Fig. 16.21). Instead, it is hydroxylated at the α-carbon by phytanoyl CoA α-hydroxylase (PhyH); carbon 1 is released as CO2; and the product, 15-carbon-long pristanal, is oxidized to pristanic acid, which is activated to its CoA derivative and undergoes β-oxidation. Refsum disease is a rare, autosomal-recessive disorder caused by a deficiency of peroxisomal PhyH. This results in the accumulation of phytanic acid in the plasma and tissues. The symptoms are primarily neurologic, and the treatment involves dietary restriction to halt disease progression. [Note: ω-Oxidation (at the methyl terminus) also is known and generates dicarboxylic acids. Normally a minor pathway of the SER, its upregulation is seen with conditions such as MCAD deficiency that limit fatty acid β-oxidation.]


Biochemistry_Lippinco

Biochemistry_Lippinco. V. KETONE BODIES: ALTERNATIVE FUEL FOR CELLS


Biochemistry_Lippinco

Biochemistry_Lippinco. Liver mitochondria have the capacity to convert acetyl CoA derived from fatty acid oxidation into ketone bodies. The compounds categorized as ketone bodies are acetoacetate, 3-hydroxybutyrate (also called β-hydroxybutyrate), and acetone (a nonmetabolized side product, Fig. 16.22). [Note: The two functional ketone bodies are organic acids.] Acetoacetate and 3-hydroxybutyrate are transported in the blood to the peripheral tissues. There they can be reconverted to acetyl CoA, which can be oxidized by the TCA cycle. Ketone bodies are important sources of energy for the peripheral tissues because they 1) are soluble in aqueous solution and, therefore, do not need to be incorporated into lipoproteins or carried by albumin as do the other lipids; 2) are produced in the liver during periods when the amount of acetyl CoA present exceeds the oxidative capacity of the liver; and 3) are used in proportion to their concentration in the blood by extrahepatic tissues, such as skeletal and cardiac


Biochemistry_Lippinco

Biochemistry_Lippinco. the amount of acetyl CoA present exceeds the oxidative capacity of the liver; and 3) are used in proportion to their concentration in the blood by extrahepatic tissues, such as skeletal and cardiac muscle, the intestinal mucosa, and the renal cortex. Even the brain can use ketone bodies to help meet its energy needs if the blood levels rise sufficiently. Thus, ketone bodies spare glucose, which is particularly important during prolonged periods of fasting (see p. 332). [Note: Disorders of fatty acid oxidation present with the general picture of hypoketosis (because of decreased availability of acetyl CoA) and hypoglycemia (because of increased reliance on glucose for energy).] carbon dioxide.


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Biochemistry_Lippinco. A. Ketone body synthesis by the liver: Ketogenesis During a fast, the liver is flooded with fatty acids mobilized from adipose tissue. The resulting elevated hepatic acetyl CoA produced by fatty acid oxidation inhibits pyruvate dehydrogenase (see p. 111) and activates pyruvate carboxylase ([PC] see p. 119). The OAA produced by PC is used by the liver for gluconeogenesis rather than for the TCA cycle. Additionally, fatty acid oxidation decreases the NAD+/NADH ratio, and the rise in NADH shifts OAA to malate (see p. 113). The decreased availability of OAA for condensation with acetyl CoA results in the increased use of acetyl CoA for ketone body synthesis. [Note: Acetyl CoA for ketogenesis is also generated by the catabolism of ketogenic amino acids (see p. 262).] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3-Hydroxy-3-methylglutaryl CoA synthesis: The first step, formation of acetoacetyl CoA, occurs by reversal of the final thiolase reaction of fatty acid oxidation (see Fig. 16.17). Mitochondrial 3-hydroxy-3methylglutaryl (HMG) CoA synthase combines a third molecule of acetyl CoA with acetoacetyl CoA to produce HMG CoA. HMG CoA synthase is the rate-limiting step in the synthesis of ketone bodies and is present in significant quantities only in the liver. [Note: HMG CoA is also an intermediate in cytosolic cholesterol synthesis (see p. 220). The two pathways are separated by location in, and conditions of, the cell.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Ketone body synthesis: HMG CoA is cleaved by HMG CoA lyase to produce acetoacetate and acetyl CoA, as shown in Figure 16.22. Acetoacetate can be reduced to form 3-hydroxybutyrate with NADH as the electron donor. [Note: Because ketone bodies are not linked to CoA, they can cross the inner mitochondrial membrane.] Acetoacetate can also spontaneously decarboxylate in the blood to form acetone, a volatile, biologically nonmetabolized compound that can be detected in the breath. The equilibrium between acetoacetate and 3-hydroxybutyrate is determined by the NAD+/NADH ratio. Because this ratio is low during fatty acid oxidation, 3-hydroxybutyrate synthesis is favored. B. Ketone body use by the peripheral tissues: Ketolysis


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Biochemistry_Lippinco. B. Ketone body use by the peripheral tissues: Ketolysis Although the liver constantly synthesizes low levels of ketone bodies, their production increases during fasting when ketone bodies are needed to provide energy to the peripheral tissues. 3-Hydroxybutyrate is oxidized to acetoacetate by 3-hydroxybutyrate dehydrogenase, producing NADH (Fig. 16.23). Acetoacetate is then provided with a CoA molecule taken from succinyl CoA by succinyl CoA:acetoacetate CoA transferase (thiophorase). This reaction is reversible, but the product, acetoacetyl CoA, is actively removed by its cleavage to two acetyl CoA by thiolase. This pulls the reaction forward. Extrahepatic tissues, including the brain but excluding cells lacking mitochondria (for example, RBC), efficiently oxidize acetoacetate and 3-hydroxybutyrate in this manner. In contrast, although the liver actively produces ketone bodies, it lacks thiophorase and, therefore, is unable to use ketone bodies as fuel.


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Biochemistry_Lippinco. C. Excessive ketone body production in diabetes mellitus When the rate of formation of ketone bodies is greater than the rate of their use, their levels begin to rise in the blood (ketonemia) and, eventually, in the urine (ketonuria). This is seen most often in cases of uncontrolled type 1 diabetes mellitus (T1D), where the blood concentration of ketone bodies may reach 90 mg/dl (versus <3 mg/dl in normal individuals), and urinary excretion of ketone bodies may be as high as 5,000 mg/24 hour. The elevation of the ketone body concentration in the blood can result in acidemia. [Note: The carboxyl group of a ketone body has a pKa of ~4.


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Biochemistry_Lippinco. Therefore, each ketone body loses a proton (H+) as it circulates in the blood, which lowers the pH.] Also, in uncontrolled T1D, urinary loss of glucose and ketone bodies results in dehydration. Therefore, the increased number of H+ circulating in a decreased volume of plasma can cause a severe acidosis (ketoacidosis, Fig. 16.24) known as diabetic ketoacidosis (DKA).] A frequent symptom of DKA is a fruity odor on the breath, which results from increased production of acetone. Ketoacidosis may also be seen in cases of prolonged fasting (see p. 330) and excessive ethanol consumption (see p. 318). VI. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. A fatty acid, generally a linear hydrocarbon chain with a terminal carboxyl group, can be saturated or unsaturated. Two unsaturated fatty acids are dietary essentials: linoleic and α-linolenic acids. Fatty acids are synthesized in the liver cytosol following a meal containing excess carbohydrate and protein. Carbons used to synthesize fatty acids are provided by acetyl coenzyme A (CoA), energy by ATP, and reducing equivalents by nicotinamide adenine dinucleotide phosphate ([NADPH], Fig. 16.25) provided by the pentose phosphate pathway and malic enzyme. Citrate carries two-carbon acetyl units from the mitochondrial matrix to the cytosol. The regulated step in fatty acid synthesis is the carboxylation of acetyl CoA to malonyl CoA by biotin-and ATP-requiring acetyl CoA carboxylase (ACC). Citrate allosterically activates ACC, and palmitoyl CoA inhibits it. ACC can also be activated by insulin and inactivated by adenosine monophosphate–activated protein kinase (AMPK) in response to


Biochemistry_Lippinco

Biochemistry_Lippinco. Citrate allosterically activates ACC, and palmitoyl CoA inhibits it. ACC can also be activated by insulin and inactivated by adenosine monophosphate–activated protein kinase (AMPK) in response to epinephrine, glucagon, or a rise in AMP. The remaining steps in fatty acid synthesis are catalyzed by the multifunctional enzyme, fatty acid synthase, which produces palmitoyl CoA by adding two-carbon units from malonyl CoA to a series of acyl acceptors. Fatty acids can be elongated and desaturated in the smooth endoplasmic reticulum (SER). When fatty acids are required for energy, hormone-sensitive lipase (activated by epinephrine, and inhibited by insulin), along with other lipases, degrades triacylglycerol (TAG) stored in adipocytes. The fatty acid products are carried by serum albumin to the liver and peripheral tissues, where their oxidation provides energy. The glycerol backbone of the degraded TAG is carried by the blood to the liver, where it serves as a gluconeogenic precursor. Fatty


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Biochemistry_Lippinco. and peripheral tissues, where their oxidation provides energy. The glycerol backbone of the degraded TAG is carried by the blood to the liver, where it serves as a gluconeogenic precursor. Fatty acid degradation (β-oxidation) occurs in mitochondria. The carnitine shuttle is required to transport long-chain fatty acids from the cytosol to the mitochondrial matrix. A translocase and the enzymes carnitine palmitoyltransferases (CPT) I and II are required. CPT-I is inhibited by malonyl CoA, thereby preventing simultaneous synthesis and degradation of fatty acids. Mitochondrial fatty acid β-oxidation produces acetyl CoA, nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FADH2). The first step in β-oxidation is catalyzed by one of four acyl CoA dehydrogenases, each with chain-length specificity. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency causes a decrease in fatty acid oxidation (process stops once a medium-chain fatty acid is produced), resulting


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Biochemistry_Lippinco. chain-length specificity. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency causes a decrease in fatty acid oxidation (process stops once a medium-chain fatty acid is produced), resulting in hypoketonemia and severe hypoglycemia. Oxidation of fatty acids with an odd number of carbons proceeds two carbons at a time (producing acetyl CoA) until three-carbon propionyl CoA remains. This compound is carboxylated to methylmalonyl CoA (by biotin-and ATP-requiring propionyl CoA carboxylase), which is then converted to succinyl CoA (a gluconeogenic precursor) by vitamin B12-requiring methylmalonyl CoA mutase. A genetic error in the mutase or vitamin B12 deficiency causes methylmalonic acidemia and aciduria. β-Oxidation of unsaturated fatty acids requires additional enzymes. β-Oxidation of verylong-chain fatty acids and α-oxidation of branched-chain fatty acids occur in the peroxisome. Deficiencies result in X-linked adrenoleukodystrophy and Refsum disease, respectively. ω-Oxidation,


Biochemistry_Lippinco

Biochemistry_Lippinco. verylong-chain fatty acids and α-oxidation of branched-chain fatty acids occur in the peroxisome. Deficiencies result in X-linked adrenoleukodystrophy and Refsum disease, respectively. ω-Oxidation, normally a minor pathway, occurs in the SER. Liver mitochondria can convert acetyl CoA derived from fatty acid oxidation into acetoacetate and 3-hydroxybutyrate (ketone bodies). Peripheral tissues possessing mitochondria can oxidize 3hydroxybutyrate to acetoacetate, which can be cleaved to two acetyl CoA, thereby producing energy for the cell. Unlike fatty acids, ketone bodies are utilized by the brain and, therefore, are important fuels during a fast. Because the liver lacks thiophorase required to degrade ketone bodies, it synthesizes them specifically for the peripheral tissues. Ketoacidosis occurs when the rate of ketone body formation is greater than the rate of use, as is seen in cases of uncontrolled type 1 diabetes mellitus.


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Biochemistry_Lippinco. tricarboxylic acid; VLDL = very-low-density lipoprotein. Choose the ONE best answer. 6.1. When oleic acid, 18:1(9), is desaturated at carbon 6 and then elongated, what is the correct representation of the product? A. 19:2(7,9) B. 20:2 (ω-6) C. 20:2(6,9) D. 20:2(8,11) Correct answer = D. Fatty acids are elongated in the smooth endoplasmic reticulum by adding two carbons at a time to the carboxylate end (carbon 1) of the molecule. This pushes the double bonds at carbon 6 and carbon 9 farther away from carbon 1. The 20:2(8,11) product is an ω-9 (n-9) fatty acid. 6.2. A 4-month-old child is being evaluated for fasting hypoglycemia. Laboratory tests at admission reveal low levels of ketone bodies (hypoketonemia), free carnitine, and long-chain acylcarnitines in the blood. Free fatty acid levels in the blood were elevated. Deficiency of which of the following would best explain these findings? A. Adipose triglyceride lipase B. Carnitine transporter


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Biochemistry_Lippinco. A. Adipose triglyceride lipase B. Carnitine transporter C. Carnitine palmitoyltransferase-I D. Long-chain fatty acid dehydrogenase Correct answer = B. A defect in the carnitine transporter (primary carnitine deficiency) would result in low levels of carnitine in the blood (as a result of increased urinary loss) and low levels in the tissues. In the liver, this decreases fatty acid oxidation and ketogenesis. Consequently, blood levels of free fatty acids rise. Deficiencies of adipose triglyceride lipase would decrease fatty acid availability. Deficiency of carnitine palmitoyltransferase I would result in elevated blood carnitine. Defects in any of the enzymes of β-oxidation would result in secondary carnitine deficiency, with a rise in acylcarnitines.


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Biochemistry_Lippinco. 6.3. A teenager, concerned about his weight, attempts to maintain a fat-free diet for a period of several weeks. If his ability to synthesize various lipids were examined, he would be found to be most deficient in his ability to synthesize: A. cholesterol. B. glycolipids. C. phospholipids. D. prostaglandins. E. triacylglycerol. Correct answer = D. Prostaglandins are synthesized from arachidonic acid. Arachidonic acid is synthesized from linoleic acid, an essential fatty acid obtained by humans from dietary lipids. The teenager would be able to synthesize all other compounds but, presumably, in somewhat decreased amounts.


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Biochemistry_Lippinco. 6.4. A 6-month-old boy was hospitalized following a seizure. History revealed that for several days prior, his appetite was decreased owing to a stomach virus. At admission, his blood glucose was 24 mg/dl (age-referenced normal is 60–100). His urine was negative for ketone bodies and positive for a variety of dicarboxylic acids. Blood carnitine levels (free and acyl bound) were normal. A tentative diagnosis of medium-chain fatty acyl coenzyme A dehydrogenase (MCAD) deficiency is made. In patients with MCAD deficiency, the fasting hypoglycemia is a consequence of: A. decreased acetyl coenzyme A production. B. decreased ability to convert acetyl coenzyme A to glucose. C. increased conversion of acetyl coenzyme A to acetoacetate. D. increased production of ATP and nicotinamide adenine dinucleotide.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. decreased ability to convert acetyl coenzyme A to glucose. C. increased conversion of acetyl coenzyme A to acetoacetate. D. increased production of ATP and nicotinamide adenine dinucleotide. Correct answer = A. Impaired oxidation of fatty acids <12 carbons in length results in decreased production of acetyl-coenzyme A (CoA), the allosteric activator of pyruvate carboxylase, a gluconeogenic enzyme, and, thus, glucose levels fall. Acetyl CoA can never be used for the net synthesis of glucose. Acetoacetate is a ketone body, and with medium-chain fatty acyl CoA dehydrogenase deficiency, ketogenesis is decreased as a result of decreased production of the substrate, acetyl CoA. Impaired fatty acid oxidation means that less ATP and nicotinamide adenine dinucleotide are made, and both are needed for gluconeogenesis.


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Biochemistry_Lippinco. 6.5. Explain why with Zellweger syndrome both very-long-chain fatty acids (VLCFA) and long-chain phytanic acid accumulate, whereas with X-linked adrenoleukodystrophy, only VLCFA accumulate. Zellweger syndrome is caused by an inability to target matrix proteins to the peroxisome. Therefore, all peroxisomal activities are affected because functional peroxisomes are unable to be formed. In X-linked adrenoleukodystrophy, the defect is an inability to transport VLCFA into the peroxisome, but other peroxisomal functions, such as α-oxidation, are normal. Phospholipid, Glycosphingolipid, and Eicosanoid Metabolism 17 For additional ancillary materials related to this chapter, please visit thePoint. I. PHOSPHOLIPID OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Phospholipids are polar, ionic compounds composed of an alcohol that is attached by a phosphodiester bond to either diacylglycerol (DAG) or sphingosine. Like fatty acids (FA), phospholipids are amphipathic in nature. That is, each has a hydrophilic head, which is the phosphate group plus whatever alcohol is attached to it (for example, serine, ethanolamine, and choline; highlighted in blue in Fig. 17.1A), and a long, hydrophobic tail containing FA or FA-derived hydrocarbons (shown in orange in Fig. 17.1A). Phospholipids are the predominant lipids of cell membranes. In membranes, the hydrophobic portion of a phospholipid molecule is associated with the nonpolar portions of other membrane constituents, such as glycolipids, proteins, and cholesterol. The hydrophilic (polar) head of the phospholipid extends outward, interacting with the intracellular or extracellular aqueous environment (see Fig. 17.1A). Membrane phospholipids also function as a reservoir for intracellular messengers,


Biochemistry_Lippinco

Biochemistry_Lippinco. extends outward, interacting with the intracellular or extracellular aqueous environment (see Fig. 17.1A). Membrane phospholipids also function as a reservoir for intracellular messengers, and, for some proteins, phospholipids serve as anchors to cell membranes. Nonmembrane phospholipids serve additional functions in the body, for example, as components of lung surfactant and essential components of bile, where their detergent properties aid cholesterol solubilization.


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Biochemistry_Lippinco. II. PHOSPHOLIPID STRUCTURE There are two classes of phospholipids: those that have glycerol (from glucose) as a backbone and those that have sphingosine (from serine and palmitate). Both classes are found as structural components of membranes, and both play a role in the generation of lipid signaling molecules. A. Glycerophospholipids Phospholipids that contain glycerol are called glycerophospholipids (or phosphoglycerides). Glycerophospholipids constitute the major class of phospholipids and are the predominant lipids in membranes. All contain (or are derivatives of) phosphatidic acid (PA), which is DAG with a phosphate group on carbon 3 (Fig. 17.1B). PA is the simplest phosphoglyceride and is the precursor of the other members of this group. 1. From phosphatidic acid and an alcohol: The phosphate group on PA can be esterified to a compound containing an alcohol group (see Fig. 17.1). For example: 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. From phosphatidic acid and an alcohol: The phosphate group on PA can be esterified to a compound containing an alcohol group (see Fig. 17.1). For example: 2. Cardiolipin: Two molecules of PA esterified through their phosphate groups to an additional molecule of glycerol form cardiolipin, or diphosphatidylglycerol (Fig. 17.2). Cardiolipin is found in membranes in bacteria and eukaryotes. In eukaryotes, cardiolipin is virtually exclusive to the inner mitochondrial membrane, where it maintains the structure and function of certain respiratory complexes of the electron transport chain. [Note: Cardiolipin is antigenic and is recognized by antibodies 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Plasmalogens: When the FA at carbon 1 of a glycerophospholipid is replaced by an unsaturated alkyl group attached by an ether (rather than by an ester) linkage to the core glycerol molecule, an ether phosphoglyceride known as a plasmalogen is produced. For example, phosphatidalethanolamine, which is abundant in nerve tissue (Fig. 17.3A), is the plasmalogen that is similar in structure to (Ab) raised against Treponema pallidum, the bacterium that causes syphilis. The Wasserman test for syphilis detects these Ab.] phosphatidylethanolamine. Phosphatidalcholine (abundant in heart muscle) is the other quantitatively significant ether lipid in mammals. [Note: Plasmalogens have “al” rather than “yl” in their names.] plasmalogen phosphatidalethanolamine. B. Platelet-activating factor. ( is a long, hydrophobic hydrocarbon chain.) 4. Platelet-activating factor: A second example of an ether glycerophospholipid is platelet-activating factor (PAF), which has a saturated alkyl group in an ether


Biochemistry_Lippinco

Biochemistry_Lippinco. hydrophobic hydrocarbon chain.) 4. Platelet-activating factor: A second example of an ether glycerophospholipid is platelet-activating factor (PAF), which has a saturated alkyl group in an ether link to carbon 1 and an acetyl residue (rather than a FA) at carbon 2 of the glycerol backbone (Fig. 17.3B). PAF is synthesized and released by a variety of cell types. It binds to surface receptors, triggering potent thrombotic and acute inflammatory events. For example, PAF activates inflammatory cells and mediates hypersensitivity, acute inflammatory, and anaphylactic reactions. It causes platelets to aggregate and activate and neutrophils and alveolar macrophages to generate superoxide radicals to kill bacteria (see p. 150). It also lowers blood pressure. [Note: PAF is one of the most potent bioactive molecules known, causing effects at concentrations as low as 10−11 mol/l.]


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Biochemistry_Lippinco. B. Sphingophospholipids: Sphingomyelin The backbone of sphingomyelin is the amino alcohol sphingosine, rather than glycerol (Fig. 17.4). A long-chain-length FA (LCFA) is attached to the amino group of sphingosine through an amide linkage, producing a ceramide, which can also serve as a precursor of glycolipids (see p. 209). The alcohol group at carbon 1 of sphingosine is esterified to phosphorylcholine, producing sphingomyelin, the only significant sphingophospholipid in humans. Sphingomyelin is an important constituent of the myelin sheath of nerve fibers. [Note: The myelin sheath is a layered, membranous structure that insulates and protects neuronal axons of the central nervous system (CNS).] III. PHOSPHOLIPID SYNTHESIS


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Biochemistry_Lippinco. Glycerophospholipid synthesis involves either the donation of PA from cytidine diphosphate (CDP)-DAG to an alcohol or the donation of the phosphomonoester of the alcohol from CDP-alcohol to DAG (Fig. 17.5). In both cases, the CDP-bound structure is considered an activated intermediate, and cytidine monophosphate (CMP) is released as a side product. Therefore, a key concept in glycerophospholipid synthesis is activation, of either DAG or the alcohol to be added, by linkage with CDP. [Note: This is similar in principle to the activation of sugars by their attachment to uridine diphosphate (UDP) (see p. 126).] The FA esterified to the glycerol alcohol groups can vary widely, contributing to the heterogeneity of this group of compounds, with saturated FA typically found at carbon 1 and unsaturated ones at carbon 2. Most phospholipids are synthesized in the smooth endoplasmic reticulum (SER). From there, they are transported to the Golgi and then to membranes of organelles or the plasma


Biochemistry_Lippinco

Biochemistry_Lippinco. ones at carbon 2. Most phospholipids are synthesized in the smooth endoplasmic reticulum (SER). From there, they are transported to the Golgi and then to membranes of organelles or the plasma membrane or are secreted from the cell by exocytosis. [Note: Ether lipid synthesis from dihydroxyacetone phosphate begins in peroxisomes.] pyrophosphate. ( is a fatty acid hydrocarbon chain.)


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Phosphatidic acid PA is the precursor of other glycerophospholipids. The steps in its synthesis from glycerol 3-phosphate and two fatty acyl coenzyme A (CoA) molecules were illustrated in Figure 16.14, p. 189, in which PA is shown as a precursor of triacylglycerol (TAG). Essentially all cells except mature erythrocytes can synthesize phospholipids, whereas TAG synthesis occurs essentially only in the liver, adipose tissue, lactating mammary glands, and intestinal mucosal cells. B. Phosphatidylcholine and phosphatidylethanolamine


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Phosphatidylcholine and phosphatidylethanolamine The neutral phospholipids PC and PE are the most abundant phospholipids in most eukaryotic cells. The primary route of their synthesis uses choline and ethanolamine obtained either from the diet or from the turnover of the body’s phospholipids. [Note: In the liver, PC also can be synthesized from PS and PE (see 2. below).] 1. Synthesis from preexisting choline and ethanolamine: These synthetic pathways involve the phosphorylation of choline or ethanolamine by kinases, followed by conversion to the activated form, CDP-choline or CDP-ethanolamine. Finally, choline phosphate or ethanolamine phosphate is transferred from the nucleotide (leaving CMP) to a molecule of DAG (see Fig. 17.5). a.


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Biochemistry_Lippinco. a. Significance of choline reutilization: The reutilization of choline is important because, although humans can synthesize choline de novo, the amount made is insufficient for our needs. Thus, choline is an essential dietary nutrient with an adequate intake (see p. 358) of 550 mg for men and 425 mg for women. [Note: Choline is also used for the synthesis of acetylcholine, a neurotransmitter.] b.


Biochemistry_Lippinco

Biochemistry_Lippinco. Phosphatidylcholine in lung surfactant: The pathway described above is the principal pathway for the synthesis of dipalmitoylphosphatidylcholine (DPPC or, dipalmitoyl lecithin). In DPPC, positions 1 and 2 on the glycerol are occupied by palmitate, a saturated LCFA. DPPC, made and secreted by type II pneumocytes, is a major lipid component of lung surfactant, which is the extracellular fluid layer lining the alveoli. Surfactant serves to decrease the surface tension of this fluid layer, reducing the pressure needed to reinflate alveoli, thereby preventing alveolar collapse (atelectasis). [Note: Surfactant is a complex mixture of lipids (90%) and proteins (10%), with DPPC being the major component for reducing surface tension.]


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Biochemistry_Lippinco. Fetal lung maturity can be gauged by determining the DPPC/sphingomyelin ratio, usually written as L (for lecithin)/S, in amniotic fluid. A value ≥2 is evidence of maturity, because it reflects the shift from sphingomyelin to DPPC synthesis that occurs in pneumocytes at ~32 weeks’ gestation.


Biochemistry_Lippinco

Biochemistry_Lippinco. c. Lung maturity: Respiratory distress syndrome (RDS) in preterm infants is associated with insufficient surfactant production and/or secretion and is a significant cause of all neonatal deaths in Western countries. Lung maturation can be accelerated by giving the mother glucocorticoids shortly before delivery to induce expression of specific genes. Postnatal administration of natural or synthetic surfactant (by intratracheal instillation) is also used. [Note: Acute RDS, seen in all age groups, is the result of alveolar damage (due to infection, injury, or aspiration) that causes fluid to accumulate in the alveoli, impeding the exchange of oxygen (O2) and carbon dioxide (CO2).] 2. Phosphatidylcholine synthesis from phosphatidylserine: The liver requires a mechanism for producing PC, even when free choline levels are low, because it exports significant amounts of PC in the bile and as a component of plasma lipoproteins. To provide the needed PC, PS is decarboxylated to PE by PS


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Biochemistry_Lippinco. even when free choline levels are low, because it exports significant amounts of PC in the bile and as a component of plasma lipoproteins. To provide the needed PC, PS is decarboxylated to PE by PS decarboxylase. PE then undergoes three methylation steps to produce PC, as illustrated in Figure 17.6. Sadenosylmethionine is the methyl group donor (see p. 264).


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Biochemistry_Lippinco. C. Phosphatidylserine PS synthesis in mammalian tissues is provided by the base exchange reaction, in which the ethanolamine of PE is exchanged for free serine (see Fig. 17.6). This reaction, although reversible, is used primarily to produce the PS required for membrane synthesis. PS has a net negative charge. (See online Chapter 35 for the role of PS in clotting.) D. Phosphatidylinositol


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Biochemistry_Lippinco. PI is synthesized from free inositol and CDP-DAG, as shown in Figure 17.5. PI is an unusual phospholipid in that it most frequently contains stearic acid on carbon 1 and arachidonic acid on carbon 2 of the glycerol. Therefore, PI serves as a reservoir of arachidonic acid in membranes and, thus, provides the substrate for prostaglandin (see p. 213) synthesis when required. Like PS, PI has a net negative charge. [Note: There is asymmetry in the phospholipid composition of the cell membrane. PS and PI, for example, are found primarily on the inner leaflet. Asymmetry is achieved by ATP-dependent enzymes known as “flippases” and “floppases.”] 1. Role in signal transduction across membranes: The phosphorylation of membrane-bound PI produces polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate ([PIP2]; Fig. 17.7). The cleavage of PIP2 by phospholipase C occurs in response to the binding of various neurotransmitters, hormones, and growth factors to G protein–coupled receptors


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Biochemistry_Lippinco. ([PIP2]; Fig. 17.7). The cleavage of PIP2 by phospholipase C occurs in response to the binding of various neurotransmitters, hormones, and growth factors to G protein–coupled receptors (GPCR), such as the α1 adrenergic receptor, on the cell membrane and activation of the Gq α-subunit (Fig. 17.8). The products of this cleavage, inositol 1,4,5-trisphosphate (IP3) and DAG, mediate the mobilization of intracellular calcium and the activation of protein kinase


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Biochemistry_Lippinco. C, which act synergistically to evoke specific cellular responses. Signal transduction across the membrane is, thus, accomplished.


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Biochemistry_Lippinco. 2. Role in membrane protein anchoring: Specific proteins can be covalently attached through a carbohydrate bridge to membrane-bound PI (Fig. 17.9). For example, lipoprotein lipase, an enzyme that degrades triacylglycerol in lipoprotein particles (see p. 228), is attached to capillary endothelial cells by a glycosyl phosphatidylinositol (GPI) anchor. [Note: GPI-linked proteins are also found in a variety of parasitic protozoans, such as trypanosomes and leishmania.] Being attached to a membrane lipid (rather than being an integral part of the membrane) allows GPI-anchored proteins increased lateral mobility on the extracellular surface of the plasma membrane. The protein can be cleaved from its anchor by the action of phospholipase C (see Fig. 17.9). [Note: A deficiency in the synthesis of GPI in hematopoietic cells results in the hemolytic disease paroxysmal nocturnal hemoglobinuria, because GPI-anchored proteins protect blood cells from complement-mediated lysis.]


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Biochemistry_Lippinco. E. Phosphatidylglycerol and cardiolipin Phosphatidylglycerol occurs in relatively large amounts in mitochondrial membranes and is a precursor of cardiolipin (diphosphatidyglycerol). It is synthesized from CDP-DAG and glycerol 3-phosphate. Cardiolipin (see Fig. 17.2) is synthesized by the transfer of DAG 3-phosphate from CDPDAG to a pre-existing molecule of phosphatidylglycerol. F. Sphingomyelin


Biochemistry_Lippinco

Biochemistry_Lippinco. F. Sphingomyelin Sphingomyelin, a sphingosine-based phospholipid, is found in cell membranes and in the myelin sheath. The synthesis of sphingomyelin is shown in Figure 17.10. Briefly, palmitoyl CoA condenses with serine, as CoA and the carboxyl group (as CO2) of serine are lost. [Note: This reaction, like the decarboxylation reactions involved in the synthesis of PE from PS and of regulators from amino acids (for example, the catecholamines from tyrosine; see p. 286), requires pyridoxal phosphate (a derivative of vitamin B6) as a coenzyme.] The product is reduced in a nicotinamide adenine dinucleotide phosphate (NADPH)-requiring reaction to sphinganine (dihydrosphingosine). The sphinganine is acylated at the amino group with one of a variety of LCFA and then desaturated to produce a ceramide, the immediate precursor of sphingomyelin (and other sphingolipids, as described on p. 208).


Biochemistry_Lippinco

Biochemistry_Lippinco. Ceramides play a key role in maintaining the skin’s water-permeability barrier. Decreased ceramide levels are associated with a number of skin diseases. Phosphorylcholine from PC is transferred to the ceramide, producing sphingomyelin and DAG. [Note: Sphingomyelin of the myelin sheath contains predominantly longer-chain FA such as lignoceric acid and nervonic acid, whereas gray matter of the brain has sphingomyelin that contains primarily stearic acid.] IV. PHOSPHOLIPID DEGRADATION The degradation of phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice. [Note: For a discussion of phospholipid digestion, see p. 175.] A number of toxins and venoms have phospholipase activity, and several pathogenic bacteria produce phospholipases that dissolve cell membranes and allow the spread of infection. Sphingomyelin is degraded by the lysosomal phospholipase, sphingomyelinase (see B. below). A. Phosphoglycerides


Biochemistry_Lippinco

Biochemistry_Lippinco. Phospholipases hydrolyze the phosphodiester bonds of phosphoglycerides, with each enzyme cleaving the phospholipid at a specific site. The major phospholipases are shown in Figure 17.11. [Note: Removal of the FA from carbon 1 or 2 of a phosphoglyceride produces a lysophosphoglyceride, which is the substrate for lysophospholipases.] Phospholipases release molecules that can serve as second messengers (for example, DAG and IP3) or that are the substrates for synthesis of messengers (for example, arachidonic acid). Phospholipases are responsible not only for degrading phospholipids but also for remodeling them. For example, phospholipases A1 and A2 remove specific FA from membrane-bound phospholipids, which can be replaced with different FA using fatty acyl CoA transferase. This mechanism is used as one way to create the unique lung surfactant DPCC (see p. 204) and to insure that carbon 2 of PI (and sometimes of PC) is bound to arachidonic acid. [Note: Barth syndrome, a rare X-linked


Biochemistry_Lippinco

Biochemistry_Lippinco. is used as one way to create the unique lung surfactant DPCC (see p. 204) and to insure that carbon 2 of PI (and sometimes of PC) is bound to arachidonic acid. [Note: Barth syndrome, a rare X-linked disorder characterized by cardiomyopathy, muscle weakness, and neutropenia, is the result of defects in cardiolipin remodeling.] phosphatidylinositol 4,5-bisphosphate; R1 and R2 = fatty acids; X = an alcohol.


Biochemistry_Lippinco

B. Sphingomyelin

Biochemistry_Lippinco. B. Sphingomyelin


Biochemistry_Lippinco

Biochemistry_Lippinco. Sphingomyelin is degraded by sphingomyelinase, a lysosomal enzyme that removes phosphorylcholine, leaving a ceramide. The ceramide is, in turn, cleaved by ceramidase into sphingosine and a free FA (Fig. 17.12). [Note: The released ceramide and sphingosine regulate signal transduction pathways, in part by influencing the activity of protein kinase C and, thus, the phosphorylation of its protein substrates. They also promote apoptosis.] Niemann-Pick disease (types A and B) is an autosomal-recessive disorder caused by the inability to degrade sphingomyelin due to a deficiency of sphingomyelinase, a type of phospholipase C. In the severe infantile form (type A, which shows <1% of normal enzymic activity), the liver and spleen are the primary sites of lipid deposits and are, therefore, greatly enlarged. The lipid consists primarily of the sphingomyelin that cannot be degraded (Fig. 17.13). Infants with this lysosomal storage disease experience rapid and progressive neurodegeneration as a


Biochemistry_Lippinco

Biochemistry_Lippinco. enlarged. The lipid consists primarily of the sphingomyelin that cannot be degraded (Fig. 17.13). Infants with this lysosomal storage disease experience rapid and progressive neurodegeneration as a result of deposition of sphingomyelin in the CNS, and they die in early childhood. A less severe variant (type B, which shows up to 10% of normal activity) with a later age of onset and a longer survival time causes little to no damage to neural tissue, but lungs, spleen, liver, and bone marrow are affected, resulting in a chronic form of the disease. Although Niemann-Pick disease occurs in all ethnic groups, type A occurs with greater frequency in the Ashkenazi Jewish population.


Biochemistry_Lippinco

Biochemistry_Lippinco. type A.] V. GLYCOLIPID OVERVIEW Glycolipids are molecules that contain both carbohydrate and lipid components. Like the phospholipid sphingomyelin, glycolipids are derivatives of ceramides in which a LCFA is attached to the amino alcohol sphingosine. Therefore, they are more precisely called glycosphingolipids. [Note: Thus, ceramides are the precursors of both phosphorylated and glycosylated sphingolipids.] Like the phospholipids, glycosphingolipids are essential components of all membranes in the body, but they are found in greatest amounts in nerve tissue. They are located in the outer leaflet of the plasma membrane, where they interact with the extracellular environment. As such, they play a role in the regulation of cellular interactions (for example, adhesion and recognition), growth, and development.


Biochemistry_Lippinco

Biochemistry_Lippinco. Membrane glycosphingolipids associate with cholesterol and GPI-anchored proteins to form lipid rafts, laterally mobile microdomains of the plasma membrane that function to organize and regulate membrane signaling and trafficking functions. Glycosphingolipids are antigenic and are the source of ABO blood group antigens (see p. 165), various embryonic antigens specific for particular stages of fetal development, and some tumor antigens. [Note: The carbohydrate portion of a glycolipid is the antigenic determinant.] They have been coopted for use as cell surface receptors for cholera and tetanus toxins as well as for certain viruses and microbes. Genetic disorders associated with an inability to properly degrade the glycosphingolipids result in lysosomal accumulation of these compounds. [Note: Changes in the carbohydrate portion of glycosphingolipids (and glycoproteins) are characteristic of transformed cells (cells with dysregulated growth).] VI. GLYCOSPHINGOLIPID STRUCTURE


Biochemistry_Lippinco

Biochemistry_Lippinco. VI. GLYCOSPHINGOLIPID STRUCTURE The glycosphingolipids differ from sphingomyelin in that they do not contain phosphate, and the polar head function is provided by a monosaccharide or oligosaccharide attached directly to the ceramide by an O-glycosidic bond (Fig. 17.14). The number and type of carbohydrate moieties present determine the type of glycosphingolipid. A. Neutral glycosphingolipids


Biochemistry_Lippinco

Biochemistry_Lippinco. The simplest neutral glycosphingolipids are the cerebrosides. These are ceramide monosaccharides that contain either a molecule of galactose (forming ceramide-galactose or galactocerebroside, the most common cerebroside found in myelin, as shown in Fig. 17.14) or glucose (forming ceramide-glucose or glucocerebroside, an intermediate in the synthesis and degradation of the more complex glycosphingolipids). [Note: Members of a group of galacto-or glucocerebrosides may also differ from each other in the type of FA attached to the sphingosine.] As their name implies, cerebrosides are found predominantly in the brain and peripheral nerves, with high concentrations in the myelin sheath. Ceramide oligosaccharides (or globosides) are produced by attaching additional monosaccharides to a glucocerebroside, for example, ceramide-glucose-galactose (also known as lactosylceramide). The additional monosaccharides can include substituted sugars such as N-acetylgalactosamine.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Acidic glycosphingolipids Acidic glycosphingolipids are negatively charged at physiologic pH. The negative charge is provided by N-acetylneuraminic acid ([NANA], a sialic acid, as shown in Fig. 17.15) in gangliosides or by sulfate groups in sulfatides. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Gangliosides: These are the most complex glycosphingolipids and are found primarily in the ganglion cells of the CNS, particularly at the nerve endings. They are derivatives of ceramide oligosaccharides and contain one or more molecules of NANA (from CMP-NANA). The notation for these compounds is G (for ganglioside) plus a subscript M, D, T, or Q to indicate whether there is one (mono), two (di), three (tri), or four (quatro) molecules of NANA in the ganglioside, respectively. Additional numbers and letters in the subscript designate the monomeric sequence of the carbohydrate attached to the ceramide. (See Fig. 17.15 for the structure of GM2.) Gangliosides are of medical interest because several lipid storage disorders involve the accumulation of NANA-containing glycosphingolipids in cells (see Fig. 17.20, p. 212). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Sulfatides: These sulfoglycosphingolipids are sulfated galactocerebrosides that are negatively charged at physiologic pH. Sulfatides are found predominantly in the brain and kidneys. VII. GLYCOSPHINGOLIPID SYNTHESIS AND DEGRADATION Synthesis of glycosphingolipids occurs primarily in the Golgi by sequential addition of glycosyl monomers transferred from UDP-sugar donors to the acceptor molecule. The mechanism is similar to that used in glycoprotein synthesis (see p. 166). A. Enzymes involved in synthesis The enzymes involved in the synthesis of glycosphingolipids are glycosyltransferases that are specific for the type and location of the glycosidic bond formed. [Note: These enzymes can recognize both glycosphingolipids and glycoproteins as substrates.] B. Sulfate group addition


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Sulfate group addition A sulfate group from the sulfate carrier 3ʹ-phosphoadenosine-5ʹphosphosulfate ([PAPS], Fig. 17.16) is added by a sulfotransferase to the 3ʹ-hydroxyl group of the galactose in a galactocerebroside, forming the sulfatide galactocerebroside 3-sulfate (Fig. 17.17). [Note: PAPS is also the sulfur donor in glycosaminoglycan synthesis (see p. 162) and steroid hormone catabolism (see p. 240).] An overview of the synthesis of sphingolipids is shown in Figure 17.18. C. Glycosphingolipid degradation


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Glycosphingolipid degradation Glycosphingolipids are internalized by phagocytosis as described for the glycosaminoglycans (see p. 163). All of the enzymes required for the degradative process are present in lysosomes, which fuse with the phagosomes. The lysosomal enzymes hydrolytically and irreversibly cleave specific bonds in the glycosphingolipid. As seen with the glycosaminoglycans and glycoproteins (see p. 170), degradation is a sequential process following the rule “last on, first off,” in which the last group added during synthesis is the first group removed in degradation. Therefore, defects in the degradation of the polysaccharide chains in these three glycoconjugates result in lysosomal storage diseases. D. Sphingolipidoses


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Sphingolipidoses In a normal individual, synthesis and degradation of glycosphingolipids are balanced, so that the amount of these compounds present in membranes is constant. If a specific lysosomal acid hydrolase required for degradation is partially or totally missing, a sphingolipid accumulates. Lysosomal lipid storage diseases caused by these deficiencies are called sphingolipidoses. The result of a specific acid hydrolase deficiency may be seen dramatically in nerve tissue, where neurologic deterioration can lead to early death. Figure 17.20 provides an outline of the pathway of sphingolipid degradation and descriptions of some sphingolipidoses. [Note: Some sphingolipidoses can also result from defects in lysosomal activator proteins (for example, the saposins) that facilitate access of the hydrolases to short carbohydrate chains as degradation proceeds.] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Common properties: A specific lysosomal hydrolytic enzyme is deficient in the classic form of each disorder. Therefore, usually, only a single sphingolipid (the substrate for the deficient enzyme) accumulates in the involved organs in each disease. [Note: The rate of biosynthesis of the accumulating lipid is normal.] The disorders are progressive and, although many are fatal in childhood, extensive phenotypic variability is seen leading to the designation of different clinical types, such as types A and B in Niemann-Pick disease. Genetic variability is also seen because a given disorder can be caused by any one of a variety of mutations within a single gene. The sphingolipidoses are autosomal-recessive disorders, except for Fabry disease, which is X linked. The incidence of the sphingolipidoses is low in most populations, except for Gaucher and Tay-Sachs diseases, which, like Niemann-Pick disease, show a high frequency in the Ashkenazi Jewish population. [Note: Tay-Sachs also has a


Biochemistry_Lippinco

Biochemistry_Lippinco. is low in most populations, except for Gaucher and Tay-Sachs diseases, which, like Niemann-Pick disease, show a high frequency in the Ashkenazi Jewish population. [Note: Tay-Sachs also has a high frequency in Irish American, French Canadian, and Louisiana Cajun populations.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Diagnosis and treatment: A specific sphingolipidosis can be diagnosed by measuring enzyme activity in cultured fibroblasts or peripheral leukocytes or by analyzing DNA (see Chapter 34). Histologic examination of the affected tissue is also useful. [Note: Shell-like inclusion bodies are seen in Tay-Sachs, and a crumpled tissue paper appearance of the cytosol is seen in Gaucher disease (Fig. 17.19).] Prenatal diagnosis, using cultured amniocytes or chorionic villi, is available. Gaucher disease, in which macrophages become engorged with glucocerebroside, and Fabry disease, in which globosides accumulate in the vascular endothelial lysosomes of the brain, heart, kidneys, and skin, are treated by recombinant human enzyme replacement therapy, but the monetary cost is extremely high. Gaucher has also been treated by bone marrow transplantation (because macrophages are derived from hematopoietic stem cells) and by substrate reduction therapy through pharmacologic reduction of


Biochemistry_Lippinco

Biochemistry_Lippinco. Gaucher has also been treated by bone marrow transplantation (because macrophages are derived from hematopoietic stem cells) and by substrate reduction therapy through pharmacologic reduction of glucosylceramide, the substrate for the deficient enzyme.


Biochemistry_Lippinco

Biochemistry_Lippinco. VIII. EICOSANOIDS: PROSTAGLANDINS, THROMBOXANES, AND LEUKOTRIENES


Biochemistry_Lippinco

Biochemistry_Lippinco. Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT) are collectively known as eicosanoids to reflect their origin from ω-3 and ω-6 polyunsaturated FA with 20 carbons (eicosa = 20). They are extremely potent compounds that elicit a wide range of responses, both physiologic (inflammatory response) and pathologic (hypersensitivity). They insure gastric integrity and renal function, regulate smooth muscle contraction (the intestine and uterus are key sites) and blood vessel diameter, and maintain platelet homeostasis. Although they have been compared to hormones in terms of their actions, eicosanoids differ from endocrine hormones in that they are produced in very small amounts in almost all tissues rather than in specialized glands and act locally rather than after transport in the blood to distant sites. Eicosanoids are not stored, and they have an extremely short half-life, being rapidly metabolized to inactive products. Their biologic actions are mediated by plasma membrane


Biochemistry_Lippinco

Biochemistry_Lippinco. blood to distant sites. Eicosanoids are not stored, and they have an extremely short half-life, being rapidly metabolized to inactive products. Their biologic actions are mediated by plasma membrane GPCR (see p. 94), which are different in different organ systems and typically result in changes in cyclic adenosine monophosphate production. Examples of eicosanoid structures are shown in Figure 17.21.


Biochemistry_Lippinco

Biochemistry_Lippinco. life. Cer = ceramide; Gal = galactose; Glc = glucose; GalNAc = Nacetylgalactosamine; NANA = N-acetylneuraminic acid; CNS = central nervous system. = sulfate; ERT = enzyme replacement therapy. known as prostacyclin. Thromboxanes are designated by TX and leukotrienes by LT.] A. Prostaglandin and thromboxane synthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Prostaglandin and thromboxane synthesis Arachidonic acid, an ω-6 FA containing 20 carbons and four double bonds (an eicosatetraenoic FA), is the immediate precursor of the predominant type of human PG (series 2 or those with two double bonds, as shown in Fig. 17.22). It is derived by the elongation and desaturation of the essential FA linoleic acid, also an ω-6 FA. Arachidonic acid is incorporated into membrane phospholipids (typically PI) at carbon 2, from which it is released by phospholipase A2 (Fig. 17.23) in response to a variety of signals, such as a rise in calcium. [Note: Series 1 PG contain one double bond and are derived from an ω-6 eicosatrienoic FA, dihomo-γ-linolenic acid, whereas series 3 PG contain three double bonds and are derived from eicosapentaenoic acid (EPA), an ω-3 FA. See p. 363.] activities (cyclooxygenase and peroxidase) of PGH2 synthase (prostaglandin endoperoxide synthase). G-SH = reduced glutathione; G-S-S-G = oxidized glutathione; PG = prostaglandin.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Prostaglandin H2 synthase: The first step in PG and TX synthesis is the oxidative cyclization of free arachidonic acid to yield PGH2 by PGH2 synthase (or, prostaglandin endoperoxide synthase). This enzyme is an ER membrane-bound protein that has two catalytic activities: fatty acid cyclooxygenase (COX), which requires two molecules of O2, and peroxidase, which requires reduced glutathione (see p. 148). PGH2 is converted to a variety of PG and TX, as shown in Figure 17.23, by cell-specific synthases. [Note: PG contain a five-carbon ring, whereas TX contain a heterocyclic six-membered oxane ring (see Fig. 17.21).] Two isozymes of PGH2 synthase, usually denoted as COX-1 and COX-2, are known. COX-1 is made constitutively in most tissues and is required for maintenance of healthy gastric tissue, renal homeostasis, and platelet aggregation. COX-2 is inducible in a limited number of tissues in response to products of activated immune and inflammatory cells. [Note: The increase in PG


Biochemistry_Lippinco

Biochemistry_Lippinco. tissue, renal homeostasis, and platelet aggregation. COX-2 is inducible in a limited number of tissues in response to products of activated immune and inflammatory cells. [Note: The increase in PG synthesis subsequent to the induction of COX-2 mediates the pain, heat, redness, and swelling of inflammation and the fever of infection.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Synthesis inhibition: The synthesis of PG and TX can be inhibited by unrelated compounds. For example, cortisol (a steroidal anti-inflammatory agent) inhibits phospholipase A2 activity (see Fig. 17.23) and, therefore, arachidonic acid, the substrate for PG and TX synthesis, is not released from membrane phospholipids. Aspirin, indomethacin, and phenylbutazone (all nonsteroidal anti-inflammatory drugs [NSAID]) inhibit both COX-1 and COX-2 and, thus, prevent the synthesis of the parent molecule, PGH2. [Note: Systemic inhibition of COX-1, with subsequent damage to the stomach and the kidneys and impaired clotting of blood, is the basis of aspirin’s toxicity.] Aspirin (but not other NSAID) also induces synthesis of lipoxins (anti-inflammatory mediators made from arachidonic acid) and resolvins and protectins (inflammationresolving mediators made from EPA). Inhibitors specific for COX-2 (the coxibs, for example, celecoxib) were designed to reduce pathologic inflammatory processes mediated


Biochemistry_Lippinco

Biochemistry_Lippinco. and protectins (inflammationresolving mediators made from EPA). Inhibitors specific for COX-2 (the coxibs, for example, celecoxib) were designed to reduce pathologic inflammatory processes mediated by COX-2 while maintaining the physiologic functions of COX-1. However, their use has been associated with increased risk of heart attacks, likely as a result of decreased PGI2 synthesis (see B. below), and some have been withdrawn from the market.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Thromboxanes and prostaglandins in platelet homeostasis


Biochemistry_Lippinco

Biochemistry_Lippinco. Thromboxane A2 (TXA2) is produced by COX-1 in activated platelets. It promotes platelet adhesion and aggregation and contraction of vascular smooth muscle, thereby promoting formation of blood clots (thrombi). (See online Chapter 35.) Prostacyclin (PGI2), produced by COX-2 in vascular endothelial cells, inhibits platelet aggregation and stimulates vasodilation and, so, impedes thrombogenesis. The opposing effects of TXA2 and PGI2 limit thrombi formation to sites of vascular injury. [Note: Aspirin has an antithrombogenic effect. It inhibits TXA2 synthesis by COX-1 in platelets and PGI2 synthesis by COX-2 in endothelial cells through irreversible acetylation of these isozymes (Fig. 17.24). COX-1 inhibition cannot be overcome in platelets, which lack nuclei. However, COX-2 inhibition can be overcome in endothelial cells because they have a nucleus and, therefore, can generate more of the enzyme. This difference is the basis of low-dose aspirin therapy used to lower the risk of stroke and


Biochemistry_Lippinco

Biochemistry_Lippinco. in endothelial cells because they have a nucleus and, therefore, can generate more of the enzyme. This difference is the basis of low-dose aspirin therapy used to lower the risk of stroke and heart attacks by decreasing formation of thrombi.]


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Leukotriene synthesis Arachidonic acid is converted to a variety of linear hydroperoxy (–OOH) acids by a separate pathway involving a family of lipoxygenases (LOX). For example, 5-LOX converts arachidonic acid to 5-hydroperoxy-6,8,11,14 eicosatetraenoic acid ([5-HPETE]; see Fig. 17.23). 5-HPETE is converted to a series of LT containing four double bonds, the nature of the final products varying according to the tissue. LT are mediators of allergic response and inflammation. Inhibitors of 5-LOX and LT-receptor antagonists are used in the treatment of asthma. [Note: LT synthesis is inhibited by cortisol and not by NSAID. Aspirin-exacerbated respiratory disease is a response to LT overproduction with NSAID use in ~10% of individuals with asthma.] IX. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Phospholipids are polar, ionic compounds composed of an alcohol (for example, choline or ethanolamine) attached by a phosphodiester bond either to diacylglycerol (DAG), producing phosphatidylcholine or phosphatidylethanolamine, or to the amino alcohol sphingosine (Fig. 17.25). Addition of a long-chain fatty acid to sphingosine produces a ceramide. Addition of phosphorylcholine produces the phospholipid sphingomyelin. Phospholipids are the predominant lipids of cell membranes. Nonmembrane phospholipids serve as components of lung surfactant and bile. Dipalmitoylphosphatidylcholine, also called dipalmitoyl lecithin, is the major lipid component of lung surfactant. Insufficient surfactant production causes respiratory distress syndrome. Phosphatidylinositol (PI) serves as a reservoir for arachidonic acid in membranes. The phosphorylation of membrane-bound PI produces phosphatidylinositol 4,5-bisphosphate (PIP2). This compound is degraded by phospholipase C in response to the binding of


Biochemistry_Lippinco

Biochemistry_Lippinco. acid in membranes. The phosphorylation of membrane-bound PI produces phosphatidylinositol 4,5-bisphosphate (PIP2). This compound is degraded by phospholipase C in response to the binding of various neurotransmitters, hormones, and growth factors to membrane G protein– coupled receptors. The products of this degradation, inositol 1,4,5trisphosphate (IP3) and DAG, mediate the mobilization of intracellular calcium and the activation of protein kinase C, which act synergistically to evoke cellular responses. Specific proteins can be covalently attached via a carbohydrate bridge to membrane-bound PI, forming a glycosyl phosphatidylinositol (GPI) anchor. A deficiency in GPI synthesis in hematopoietic cells results in the hemolytic disease paroxysmal nocturnal hemoglobinuria. The degradation of phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice. Sphingomyelin is degraded to a ceramide plus phosphorylcholine by the lysosomal enzyme sphingomyelinase, a


Biochemistry_Lippinco

Biochemistry_Lippinco. phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice. Sphingomyelin is degraded to a ceramide plus phosphorylcholine by the lysosomal enzyme sphingomyelinase, a deficiency of which causes Niemann-Pick (A and B) disease. Glycosphingolipids are derivatives of ceramides to which carbohydrates have been attached. Adding one sugar molecule to the ceramide produces a cerebroside, adding an oligosaccharide produces a globoside, and adding an acidic N-acetylneuraminic acid molecule produces a ganglioside. Glycosphingolipids are found predominantly in cell membranes of the brain and peripheral nervous tissue, with high concentrations in the myelin sheath. They are antigenic. Glycolipids are degraded in the lysosomes by acid hydrolases. A deficiency of any one of these enzymes causes a sphingolipidosis, in which a characteristic sphingolipid accumulates. Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT), the eicosanoids, are produced in very


Biochemistry_Lippinco

Biochemistry_Lippinco. of these enzymes causes a sphingolipidosis, in which a characteristic sphingolipid accumulates. Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT), the eicosanoids, are produced in very small amounts in almost all tissues, act locally, and have an extremely short half-life. They serve as mediators of the inflammatory response. Arachidonic acid is the immediate precursor of the predominant class of human PG (those with two double bonds). It is derived by the elongation and desaturation of the essential fatty acid linoleic acid and is stored in the membrane as a component of a phospholipid, generally PI. Arachidonic acid is released from the phospholipid by phospholipase A2 (inhibited by cortisol). Synthesis of the PG and TX begins with the oxidative cyclization of free arachidonic acid to yield PGH2 by PGH2 synthase (or, prostaglandin endoperoxide synthase), an endoplasmic reticular membrane protein that has two catalytic activities: fatty acid cyclooxygenase (COX) and


Biochemistry_Lippinco

Biochemistry_Lippinco. acid to yield PGH2 by PGH2 synthase (or, prostaglandin endoperoxide synthase), an endoplasmic reticular membrane protein that has two catalytic activities: fatty acid cyclooxygenase (COX) and peroxidase. There are two isozymes of PGH2 synthase: COX-1 (constitutive) and COX-2 (inducible). Aspirin irreversibly inhibits both.


Biochemistry_Lippinco

Biochemistry_Lippinco. Opposing effects of PGI2 and TXA2 limit clot formation. LT are linear molecules produced from arachidonic acid by the 5-lipoxygenase (5-LOX) pathway. They mediate allergic response. Their synthesis is inhibited by cortisol and not by aspirin. glycosphingolipids, and eicosanoids. PLA2 = phospholipase A2; = sulfate ion; NSAID = nonsteroidal anti-inflammatory drugs. Choose the ONE best answer. 7.1. Aspirin-exacerbated respiratory disease (AERD) is a severe reaction to nonsteroidal anti-inflammatory drugs (NSAID) characterized by bronchoconstriction 30 minutes to several hours after ingestion. Which of the following statements best explains the symptoms seen in patients with AERD? NSAID: A. inhibit the activity of the cystic fibrosis transmembrane conductance regulator protein, resulting in thickened mucus that block airways. B. inhibit cyclooxygenase but not lipoxygenase, resulting in the flow of arachidonic acid to leukotriene synthesis.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. inhibit cyclooxygenase but not lipoxygenase, resulting in the flow of arachidonic acid to leukotriene synthesis. C. activate the cyclooxygenase activity of prostaglandin H2 synthase, resulting in increased synthesis of prostaglandins that promote vasodilation. D. activate phospholipases, resulting in decreased amounts of dipalmitoylphosphatidylcholine and alveolar collapse (atelectasis). Correct answer = B. NSAID inhibit cyclooxygenase but not lipoxygenase, so any arachidonic acid available is used for the synthesis of bronchoconstricting leukotrienes. NSAID have no effect on the cystic fibrosis (CF) transmembrane conductance regulator protein, defects in which are the cause of CF. Steroids, not NSAID, inhibit phospholipase A2. Cyclooxygenase is inhibited by NSAID, not activated. NSAID have no effect on phospholipases.


Biochemistry_Lippinco

Biochemistry_Lippinco. 7.2. An infant, born at 28 weeks’ gestation, rapidly gave evidence of respiratory distress. Clinical laboratory and imaging results supported the diagnosis of infant respiratory distress syndrome. Which of the following statements about this syndrome is true? A. It is unrelated to the baby’s premature birth. B. It is a consequence of too few type II pneumocytes. C. The lecithin/sphingomyelin ratio in the amniotic fluid is likely to be high (>2). D. The concentration of dipalmitoylphosphatidylcholine in the amniotic fluid would be expected to be lower than that of a full-term baby. E. It is an easily treated disorder with low mortality. F. It is treated by administering surfactant to the mother just before she gives birth.


Biochemistry_Lippinco

Biochemistry_Lippinco. E. It is an easily treated disorder with low mortality. F. It is treated by administering surfactant to the mother just before she gives birth. Correct answer = D. Dipalmitoylphosphatidylcholine (DPPC or, dipalmitoyl lecithin) is the lung surfactant found in mature, healthy lungs. Respiratory distress syndrome (RDS) can occur in lungs that make too little of this compound. If the lecithin/sphingomyelin (L/S) ratio in amniotic fluid is ≥2, a newborn’s lungs are considered to be sufficiently mature (premature lungs would be expected to have a ratio <2). The RDS would not be due to too few type II pneumocytes, which would simply be secreting sphingomyelin rather than DPPC at 28 weeks’ gestation. The mother is given a glucocorticoid, not surfactant, prior to giving birth (antenatally). Surfactant would be administered to the baby postnatally to reduce surface tension.


Biochemistry_Lippinco

Biochemistry_Lippinco. 7.3. A 10-year-old boy was evaluated for burning sensations in his feet and clusters of small, red-purple spots on his skin. Laboratory studies revealed protein in his urine. Enzymatic analysis revealed a deficiency of αgalactosidase, and enzyme replacement therapy was recommended. The most likely diagnosis is: A. Fabry disease. B. Farber disease. C. Gaucher disease. D. Krabbe disease. E. Niemann-Pick disease. Correct answer = A. Fabry disease, a deficiency of α-galactosidase, is the only X-linked sphingolipidosis. It is characterized by pain in the extremities, a red-purple skin rash (generalized angiokeratomas), and kidney and cardiac complications. Protein in his urine indicates kidney damage. Enzyme replacement therapy is available. 7.4. Current medical advice for individuals experiencing chest pain is to call emergency medical services and chew a regular strength, noncoated aspirin. What is the basis for recommending aspirin?


Biochemistry_Lippinco

Biochemistry_Lippinco. 7.4. Current medical advice for individuals experiencing chest pain is to call emergency medical services and chew a regular strength, noncoated aspirin. What is the basis for recommending aspirin? Aspirin has an antithrombogenic effect: It prevents formation of blood clots that could occlude heart vessels. Aspirin inhibits thromboxane A2 synthesis by cyclooxygenase-1 in platelets through irreversible acetylation, thereby inhibiting platelet activation and vasoconstriction. Chewing a noncoated aspirin increases the rate of its absorption. Cholesterol, Lipoprotein, and Steroid Metabolism 18 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW