Biochemistry_Lippinco

Biochemistry_Lippinco. Genetic differences (genotypes) in the gene for catalytic subunit 1 of VKOR (VKORC1) influence patient response to warfarin. For example, a polymorphism (see p. 491) in the promoter region of the gene decreases gene expression, resulting in less VKOR being made, thereby necessitating a lower dose of warfarin to achieve a therapeutic level. Polymorphisms in the cytochrome P450 enzyme (CYP2C9) that metabolizes warfarin are also known. In 2010, the U.S. Food and Drug Administration added a genotype-based dose table to the warfarin label (package insert). The influence of genetics on an individual’s response to drugs is known as pharmacogenetics. D. Pathways Three distinct pathways are involved in formation of the fibrin meshwork: the extrinsic pathway, the intrinsic pathway, and the common pathway. Production of FXa by the extrinsic and intrinsic pathways initiates the common pathway (see Fig. 35.2).


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Extrinsic: This pathway involves a protein, tissue factor (TF), that is not in the blood but becomes exposed when blood vessels get injured. TF (or, FIII) is a transmembrane glycoprotein abundant in vascular subendothelium. It is an extravascular accessory protein and not a protease. Any injury that exposes FIII to blood rapidly (within seconds) initiates the extrinsic (or, TF) pathway. Once exposed, TF binds a circulating Gla-containing protein, FVII, activating it through conformational change. [Note: FVII can also be activated proteolytically by thrombin (see 3. below).] Binding of FVII to TF requires the presence of Ca2+ and phospholipids. The TF–FVIIa complex then binds and activates FX by proteolysis (Fig. 35.8). Therefore, activation of FX by the extrinsic pathway occurs in association with the cell membrane. The extrinsic pathway is quickly inactivated by tissue factor pathway inhibitor (TFPI) that, in a FXa-dependent process, binds to the TF–FVIIa complex and prevents


Biochemistry_Lippinco

Biochemistry_Lippinco. association with the cell membrane. The extrinsic pathway is quickly inactivated by tissue factor pathway inhibitor (TFPI) that, in a FXa-dependent process, binds to the TF–FVIIa complex and prevents further production of FXa. [Note: TF and FVII are unique to the extrinsic pathway.] 2. Intrinsic: All of the protein factors involved in the intrinsic pathway are present in the blood and are, therefore, intravascular. The intrinsic pathway involves two phases: the contact phase and the FX-activation phase, each with known deficiencies.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Contact phase: This phase results in the activation of FXII (Hageman factor) by conformational change through binding to a negative surface. Deficiencies in FXII (or in the other proteins of this phase, high molecular weight kininogen and prekallikrein) do not result in bleeding, calling into question the importance of this phase in coagulation. However, the contact phase does play a role in inflammation. [Note: FXII can be activated proteolytically by thrombin (see 3. below)]. b.


Biochemistry_Lippinco

Biochemistry_Lippinco. b. Factor X–activation phase: The sequence of events leading to the activation of FX to FXa by the intrinsic pathway is initiated by FXIIa (Fig. 35.9). FXIIa activates FXI, and FXIa activates FIX, a Glacontaining serine protease. FIXa combines with FVIIIa (a bloodborne accessory protein), and the complex activates FX, a Gla-containing serine protease. [Note: The complex containing FIXa, FVIIIa, and FX forms on exposed negatively charged membrane regions, and FX gets activated to FXa. This complex is sometimes referred to as Xase. Binding of the complex to membrane phospholipids requires Ca2+.] c. Factor XII deficiency: A deficiency in FXII does not lead to a bleeding disorder. This is because FXI, the next protein in the cascade, can be activated proteolytically by thrombin (see 3. below). d.


Biochemistry_Lippinco

Biochemistry_Lippinco. d. Hemophilia: Hemophilia is a coagulopathy, a defect in the ability to clot. Hemophilia A, which accounts for 80% of all hemophilia, results from deficiency of FVIII, whereas deficiency of FIX results in hemophilia B. Each deficiency is characterized by decreased and delayed ability to clot and/or formation of abnormally friable (easily disrupted) clots. This can be manifested, for example, by bleeding into the joints (Fig. 35.10). The extent of the factor deficiency determines the severity of the disease. Current treatment for hemophilia is factor replacement therapy using FVIII or FIX obtained from pooled human blood or from recombinant DNA technology. However, antibodies to the factors can develop. Gene therapy is a goal. Because the genes for both proteins are on the X chromosome, hemophilia is an X-linked disorder. [Note: Deficiency of FXI results in a bleeding disorder that sometimes is referred to as hemophilia C.] with hemophilia.


Biochemistry_Lippinco

Biochemistry_Lippinco. The inactivation of the extrinsic pathway by TFPI results in dependence on the intrinsic pathway for continued production of FXa. This explains why individuals with hemophilia bleed even though they have an intact extrinsic pathway.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Common: FXa produced by both the intrinsic and the extrinsic paths initiates the common pathway, a sequence of reactions that results in the generation of fibrin (FIa), as shown in Figure 35.11. FXa associates with FVa (a bloodborne accessory protein) and, in the presence of Ca2+ and phospholipids, forms a membrane-bound complex referred to as prothrombinase. The complex cleaves prothrombin (FII) to thrombin (FIIa). [Note: FVa potentiates the proteolytic activity of FXa.] The binding of Ca2+ to the Gla residues in FII facilitates the binding of FII to the membrane and to the prothrombinase complex, with subsequent cleavage to FIIa. Cleavage excises the Gla-containing region, releasing FIIa from the membrane and, thereby, freeing it to activate fibrinogen (FI) in the blood. [Note: This is the only example of cleavage of a Gla protein that results in the release of a Gla-containing peptide. The peptide travels to the liver where it is thought to act as a signal for increased


Biochemistry_Lippinco

Biochemistry_Lippinco. This is the only example of cleavage of a Gla protein that results in the release of a Gla-containing peptide. The peptide travels to the liver where it is thought to act as a signal for increased production of clotting proteins.] Oral, direct inhibitors of FXa have been approved for clinical use as anticoagulants. In contrast to warfarin, they have a more rapid onset and shorter half-life and do not require routine monitoring.


Biochemistry_Lippinco

Biochemistry_Lippinco. A common point mutation (G20210A) in which an adenine (A) replaces a guanine (G) at nucleotide 20210 in the 3′ untranslated region of the gene for FII leads to increased levels of FII in the blood. This results in thrombophilia, a condition characterized by an increased tendency for blood to clot. a. Fibrinogen cleavage to fibrin: FI is a soluble glycoprotein made by the liver. It consists of dimers of three different polypeptide chains [(αβγ)2] held together at the N termini by disulfide bonds. The N termini of the α and β chains form “tufts” on the central of three globular domains (Fig. 35.12). The tufts are negatively charged and result in repulsion between FI molecules. Thrombin (FIIa) cleaves the charged tufts (releasing fibrinopeptides A and B), and FI becomes FIa. As a result of the loss of charge, the FIa monomers are able to noncovalently associate in a staggered array, and a soft (soluble) fibrin clot is formed. b.


Biochemistry_Lippinco

Biochemistry_Lippinco. b. Fibrin cross-linking: The associated FIa molecules get covalently cross-linked. This converts the soft clot to a hard (insoluble) clot. FXIIIa, a transglutaminase, covalently links the γ-carboxamide of a glutamine residue in one FIa molecule to the ε-amino of a lysine residue in another through formation of an isopeptide bond and release of ammonia (Fig. 35.13). [Note: FXIII is also activated by thrombin.] c.


Biochemistry_Lippinco

Biochemistry_Lippinco. Importance of thrombin: The activation of FX by the extrinsic pathway provides the “spark” of FXa that results in the initial activation of thrombin. FIIa then activates factors of the common (FV, FI, FXIII), intrinsic (FXI, FVIII), and extrinsic (FVII) pathways (Fig. 35.14). It also activates FXII of the contact phase. The extrinsic pathway, then, initiates clotting by the generation of FXa, and the intrinsic pathway amplifies and sustains clotting after the extrinsic pathway has been inhibited by TFPI. [Note: Hirudin, a peptide secreted from the salivary gland of medicinal leeches, is a potent direct thrombin inhibitor (DTI).


Biochemistry_Lippinco

Biochemistry_Lippinco. Injectable recombinant hirudin has been approved for clinical use. Dabigatran is an oral DTI.] Additional crosstalk between the pathways of clotting is achieved by the FVIIa–TF-mediated activation of the intrinsic pathway and the FXIIa-mediated activation of the extrinsic pathway. The complete picture of physiologic blood clotting via the formation of a hard fibrin clot is shown in Figure 35.15. The factors of the clotting cascade are shown organized by function in Figure 35.16. activated form would be denoted by an “a” after the numeral. [Note: Calcium is IV. There is no VI. I (fibrin) is neither a protease nor an accessory protein. XIII is a transglutaminase.] Gla = γ-carboxyglutamate.


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. There is no VI. I (fibrin) is neither a protease nor an accessory protein. XIII is a transglutaminase.] Gla = γ-carboxyglutamate. Clinical laboratory tests are available to evaluate the extrinsic through common pathways (prothrombin time [PT] using thromboplastin and expressed as the international normalized ratio [INR]) and the intrinsic through common pathways (activated partial thromboplastin time [aPTT]). Thromboplastin is a combination of phospholipids + FIII. A derivative, partial thromboplastin contains just the phospholipid portion because FIII is not needed to activate the intrinsic pathway. III. LIMITING CLOTTING


Biochemistry_Lippinco

Biochemistry_Lippinco. III. LIMITING CLOTTING The ability to limit clotting to areas of damage (anticoagulation) and to remove clots once repair processes are underway (fibrinolysis) are exceedingly important aspects of hemostasis. These actions are performed by proteins that inactivate clotting factors either by binding to them and removing them from the blood or by degrading them and also by proteins that degrade the fibrin meshwork. A. Inactivating proteins Proteins synthesized by the liver and by the blood vessels themselves balance the need to form clots at sites of vessel injury with the need to limit their formation beyond the injured area.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Antithrombin: Antithrombin III (ATIII), also referred to simply as antithrombin (AT), is a hepatic protein that circulates in the blood. It inactivates free FIIa by binding to it and carrying it to the liver (Fig. 35.17). Thus, ATIII removes FIIa from the blood, preventing it from participating in coagulation. [Note: ATIII is a serine protease inhibitor, or “serpin.” A serpin contains a reactive loop to which a specific protease binds. Once bound, the protease cleaves a peptide bond in the serpin causing a conformational change that traps the enzyme in a covalent complex. α1-Antitrypsin (see p. 50) is also a serpin.] The affinity of ATIII for FIIA is greatly increased when ATIII is bound to heparin, an intracellular glycosaminogly-can (see p. 159) released in response to injury by mast cells associated with blood vessels. Heparin, an anticoagulant, is used therapeutically to limit clot formation. [Note: In contrast to the anticoagulant warfarin, which has a slow onset and a long


Biochemistry_Lippinco

Biochemistry_Lippinco. cells associated with blood vessels. Heparin, an anticoagulant, is used therapeutically to limit clot formation. [Note: In contrast to the anticoagulant warfarin, which has a slow onset and a long half-life and is administered orally, heparin has a rapid onset and a short half-life and requires intravenous administration. The two drugs are commonly used in an overlapping manner in the treatment (and prevention) of thrombosis.] ATIII also inactivates FXa and the other serine proteases of clotting, FIXa, FXIa, FXIIa, and the FVIIa–TF complex. [Note: ATIII binds to a specific pentasaccharide within the oligosaccharide form of heparin. Inhibition of FIIa requires the oligosaccharide form, whereas inhibition of FXa requires only the pentasaccharide form. Fondaparinux, a synthetic version of the pentasaccharide, is used clinically to inhibit FXa.] 2. Protein C–protein S complex: Protein C, a circulating Gla-containing protein made in the liver, is activated by FIIa complexed with


Biochemistry_Lippinco

Biochemistry_Lippinco. of the pentasaccharide, is used clinically to inhibit FXa.] 2. Protein C–protein S complex: Protein C, a circulating Gla-containing protein made in the liver, is activated by FIIa complexed with thrombomodulin. Thrombomodulin, an integral membrane glycoprotein of endothelial cells, binds FIIa, thereby decreasing FIIa’s affinity for fibrinogen and increasing its affinity for protein C. Protein C in complex with protein S, also a Gla-containing protein, forms the activated protein C (APC) complex that cleaves the accessory proteins FVa and FVIIIa, which are required for maximal activity of FXa (Fig. 35.18). Protein S helps anchor APC to the clot. Thrombomodulin, then, modulates the activity of thrombin, converting it from a protein of coagulation to a protein of anticoagulation, thereby limiting the extent of clotting. Factor V Leiden is a mutant form of FV (glutamine is substituted for arginine at position 506) that is resistant to APC. It is the most common inherited cause of


Biochemistry_Lippinco

Biochemistry_Lippinco. limiting the extent of clotting. Factor V Leiden is a mutant form of FV (glutamine is substituted for arginine at position 506) that is resistant to APC. It is the most common inherited cause of thrombophilia in the United States, with highest frequency in the Caucasian population. Heterozygotes have a 7-fold increase in the risk for venous thrombosis, and homozygotes have up to a 50-fold increase. [Note: Women with FV Leiden are at even greater risk of thrombosis during pregnancy or when taking estrogen.]


Biochemistry_Lippinco

Biochemistry_Lippinco. Thrombophilia (hypercoagulability) can result from deficiencies of proteins C, S, and ATIII; from the presence of FV Leiden and antiphospholipid antibodies; and from excess production of FII (G20210A mutation). [Note: A thrombus that forms in the deep veins of the leg (deep venous thrombosis, or DVT) can cause a pulmonary embolism (PE) if the clot (or a piece of it) breaks off, travels to the lungs, and blocks circulation.] B. Fibrinolysis


Biochemistry_Lippinco

Biochemistry_Lippinco. Clots are temporary patches that must be removed once wound repair has begun. The fibrin clot is cleaved by the protein plasmin to fibrin degradation products (Fig. 35.19). [Note: Measurement of D-dimer, a fibrin degradation product containing two cross-linked D domains released by the action of plasmin, can be used to assess the extent of clotting (see Fig. 35.12).] Plasmin is a serine protease that is generated from plasminogen by plasminogen activators. Plasminogen, secreted by the liver into the circulation, binds to FIa and is incorporated into clots as they form. Tissue plasminogen activator (TPA, t-PA), made by vascular endothelial cells and secreted in an inactive form in response to FIIa, becomes active when bound to FIa–plasminogen. Bound plasmin and TPAa are protected from their inhibitors, α2-antiplasmin and plasminogen activator inhibitors, respectively. Once the fibrin clot is dissolved, plasmin and TPAa become available to their inhibitors. Therapeutic fibrinolysis in


Biochemistry_Lippinco

Biochemistry_Lippinco. inhibitors, α2-antiplasmin and plasminogen activator inhibitors, respectively. Once the fibrin clot is dissolved, plasmin and TPAa become available to their inhibitors. Therapeutic fibrinolysis in patients with an MI or an ischemic stroke can be achieved by treatment with commercially available TPA made by recombinant DNA techniques. Mechanical clot removal (thrombectomy) is also possible. [Note: Urokinase is a plasminogen activator (u-PA) made in a variety of tissues and originally isolated from urine. Streptokinase (from bacteria) activates both free and fibrin-bound plasminogen.]


Biochemistry_Lippinco

Biochemistry_Lippinco. Plasminogen contains structural motifs known as “kringle domains” that mediate protein–protein interactions. Because lipoprotein (a) [Lp(a)] also contains kringle domains, it competes with plasminogen for binding to FIa. The potential to inhibit fibrinolysis may be the basis for the association of elevated Lp(a) with increased risk for cardiovascular disease (see p. 236). IV. PLATELET PLUG FORMATION Platelets (thrombocytes) are small, anucleate fragments of megakaryocytes that adhere to exposed collagen of damaged endothelium, get activated, and aggregate to form a platelet plug (Fig. 35.20; also see Fig. 35.1). Formation of the platelet plug is referred to as primary hemostasis because it is the first response to bleeding. In a normal adult, there are 150,000–450,000 platelets per µl of blood. They have a life span of up to 10 days, after which they are taken up by the liver and spleen and destroyed. Clinical laboratory tests to measure platelet number and activity are available.


Biochemistry_Lippinco

A. Adhesion

Biochemistry_Lippinco. A. Adhesion


Biochemistry_Lippinco

Biochemistry_Lippinco. Adhesion of platelets to exposed collagen at the site of vessel injury is mediated by the protein von Willebrand factor (VWF). VWF binds to collagen, and platelets bind to VWF via glycoprotein Ib (GPIb), a component of a membrane receptor complex (GPIb–V–IX) on the platelet surface (Fig. 35.21). Binding to VWF stops the forward movement of platelets. [Note: Deficiency in the receptor for VWF results in Bernard-Soulier syndrome, a disorder of decreased platelet adhesion.] VWF is a glycoprotein that is released from platelets. It also is made and secreted by endothelial cells. In addition to mediating the binding of platelets to collagen, VWF also binds to and stabilizes FVIII in the blood. Deficiency of VWF results in von Willebrand disease (VWD), the most common inherited coagulopathy. VWD results from decreased binding of platelets to collagen and a deficiency in FVIII (due to increased degradation). Platelets can also bind directly to collagen via the membrane receptor glycoprotein


Biochemistry_Lippinco

Biochemistry_Lippinco. results from decreased binding of platelets to collagen and a deficiency in FVIII (due to increased degradation). Platelets can also bind directly to collagen via the membrane receptor glycoprotein VI (GPVI). Once adhered, platelets get activated. [Note: Damage to the endothelium also exposes FIII, initiating the extrinsic pathway of blood clotting and activation of FX (see Fig. 35.8).]


Biochemistry_Lippinco

B. Activation

Biochemistry_Lippinco. B. Activation


Biochemistry_Lippinco

Biochemistry_Lippinco. Once adhered to areas of injury, platelets get activated. Platelet activation involves morphologic (shape) changes and degranulation, the process by which platelets secrete the contents of their α and δ (or, dense) storage granules. Activated platelets also expose PS on their surface. The externalization of PS is mediated by a Ca2+-activated enzyme known as scramblase that disrupts the membrane asymmetry created by flippases (see p. 205). Thrombin is the most potent platelet activator. FIIa binds to and activates protease-activated receptors, a type of G protein–coupled receptor (GPCR), on the surface of platelets (Fig. 35.22). FIIa is primarily associated with Gq proteins (see p. 205), resulting in activation of phospholipase C and a rise in diacylglycerol (DAG) and inositol trisphosphate (IP3). [Note: Thrombomodulin, through its binding of FIIa, decreases the availability of FIIa for platelet activation (see Fig. 35.18).] 1. Degranulation: DAG activates protein kinase C, a key event


Biochemistry_Lippinco

Biochemistry_Lippinco. (IP3). [Note: Thrombomodulin, through its binding of FIIa, decreases the availability of FIIa for platelet activation (see Fig. 35.18).] 1. Degranulation: DAG activates protein kinase C, a key event for degranulation. IP3 causes the release of Ca2+ (from dense granules). The Ca2+ activates phospholipase A2, which cleaves membrane phospholipids to release arachidonic acid, the substrate for the synthesis of thromboxane A2 (TXA2) in activated platelets by cyclooxygenase-1 (COX-1) (see p. 214). TXA2 causes vasoconstriction, augments degranulation, and binds to platelet GPCR, causing activation of additional platelets. Recall that aspirin irreversibly inhibits COX and, consequently, TXA2 synthesis and is referred to as an antiplatelet drug.


Biochemistry_Lippinco

Biochemistry_Lippinco. Degranulation also results in release of serotonin and adenosine diphosphate (ADP) from dense granules. Serotonin causes vasoconstriction. ADP binds to GPCR on the surface of platelets, activating additional platelets. [Note: Some antiplatelet drugs, such as clopidogrel, are ADP-receptor antagonists.] Platelet-derived growth factor (involved in wound healing), VWF, FV, FXIII, and FI are among other proteins released from α granules. [Note: Platelet-activating factor (PAF), an ether phospholipid (see p. 202) synthesized by a variety of cell types including endothelial cells and platelets, binds PAF receptors (GPCR) on the surface of platelets and activates them.] 2. Morphologic change: The change in shape of activated platelets from discoidal to spherical with pseudopod-like processes that facilitate platelet–platelet and platelet–surface interactions (Fig. 35.23) is initiated by the release of Ca2+ from dense granules. Ca2+ bound to calmodulin (see p. 133) mediates the activation of


Biochemistry_Lippinco

Biochemistry_Lippinco. facilitate platelet–platelet and platelet–surface interactions (Fig. 35.23) is initiated by the release of Ca2+ from dense granules. Ca2+ bound to calmodulin (see p. 133) mediates the activation of myosin light chain kinase that phosphorylates the myosin light chain, resulting in a major reorganization of the platelet cytoskeleton.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Aggregation Activation causes dramatic changes in platelets that lead to their aggregation. Structural changes in a surface receptor (GPIIb/IIIa) expose binding sites for fibrinogen. Bound FI molecules link activated platelets to one another (Fig. 35.24), with a single FI able to bind two platelets. FI is converted to FIa by FIIa and then covalently cross-linked by FXIIIa coming from both the blood and the platelets. [Note: The exposure of PS on the surface of activated platelets allows formation of the Xase complex (VIIIa, IXa, X, and Ca2+) with subsequent formation of FXa and generation of FIIa.] Fibrin formation (secondary hemostasis) strengthens the platelet plug. [Note: Rare defects in the platelet receptor for FI result in Glanzmann thrombasthenia (decreased platelet function), whereas autoantibodies to this receptor are a cause of immune thrombocytopenia (decreased platelet number).]


Biochemistry_Lippinco

Biochemistry_Lippinco. Unnecessary activation of platelets is prevented because 1) an intact vascular wall is separated from the blood by a monolayer of endothelial cells, preventing the contact of platelets with collagen; 2) endothelial cells synthesize prostaglandin I2 (PGI2, or prostacyclin) and nitric oxide, each of which causes vasodilation; and 3) endothelial cells have a cell surface ADPase that converts ADP to adenosine monophosphate. V. CHAPTER SUMMARY Blood clotting (coagulation) is designed to rapidly stop bleeding from a damaged blood vessel in order to maintain a constant blood volume (hemostasis). Coagulation is accomplished through formation of a clot (thrombus) consisting of a plug of platelets and a meshwork of the protein fibrin (Fig. 35.25).


Biochemistry_Lippinco

Biochemistry_Lippinco. The formation of the fibrin meshwork by the clotting cascade involves the extrinsic and intrinsic pathways (and their associated protein factors [F]) that converge at FXa to form the common pathway. Many of the protein factors are serine proteases with trypsin-like specificity. Calcium binds the negatively charged γ-carboxyglutamate (Gla) residues present in certain of the clotting proteases (FII, FVII, FIX, and FX), facilitating the binding of these proteins to exposed negatively charged phosphatidylserine at the site of injury and on the surface of platelets. γ-Glutamyl carboxylase and its coenzyme, the hydroquinone form of vitamin K, are required for formation of Gla residues. In the reaction, vitamin K gets oxidized to the nonfunctional epoxide form. Warfarin, a synthetic analog of vitamin K used clinically to reduce clotting, inhibits the enzyme vitamin K epoxide reductase that regenerates the functional reduced form. The extrinsic pathway is initiated by exposure of FIII (tissue


Biochemistry_Lippinco

Biochemistry_Lippinco. K used clinically to reduce clotting, inhibits the enzyme vitamin K epoxide reductase that regenerates the functional reduced form. The extrinsic pathway is initiated by exposure of FIII (tissue factor [TF]), an accessory protein, in vascular subendothelium. Exposed TF binds a circulating Glacontaining protein, FVII, activating it through conformational change. The TF–FVIIa complex then binds and activates FX by proteolysis. FXa from the extrinsic pathway allows thrombin production by the common pathway. Thrombin then activates components of the intrinsic pathway. The extrinsic pathway is rapidly inhibited by tissue factor pathway inhibitor. The intrinsic pathway is initiated by FXIIa. FXIIa activates FXI, and FXIa activates FIX. FIXa combines with FVIIIa (an accessory protein), and the complex activates FX. FVIII deficiency results in hemophilia A, whereas FIX deficiency results in the less common hemophilia B. FXa associates with FVa (an accessory protein), forming prothrombinase


Biochemistry_Lippinco

Biochemistry_Lippinco. complex activates FX. FVIII deficiency results in hemophilia A, whereas FIX deficiency results in the less common hemophilia B. FXa associates with FVa (an accessory protein), forming prothrombinase that cleaves prothrombin (FII) to thrombin (FIIa). Thrombin then cleaves fibrinogen to fibrin (FIa). Fibrin monomers associate, forming a soluble (soft) fibrin clot. The fibrin molecules get cross-linked by FXIIIa, a transglutaminase, forming an insoluble (hard) fibrin clot. Proteins synthesized by the liver and by blood vessels themselves balance coagulation with anticoagulation. Antithrombin III, a serine protease inhibitor, or serpin, binds to and removes thrombin from the blood. Its affinity for thrombin is increased by heparin, which is used therapeutically to limit clot formation. Protein C, a Gla-containing protein, is activated by the thrombin–thrombomodulin complex. Thrombomodulin decreases thrombin’s affinity for fibrinogen, converting it from a protein of coagulation to a


Biochemistry_Lippinco

Biochemistry_Lippinco. Protein C, a Gla-containing protein, is activated by the thrombin–thrombomodulin complex. Thrombomodulin decreases thrombin’s affinity for fibrinogen, converting it from a protein of coagulation to a protein of anticoagulation. Protein C in complex with protein S (a Gla-containing protein) forms the activated protein C (APC) complex that cleaves the accessory proteins FVa and FVIIIa. FV Leiden is resistant to APC. It is the most common inherited thrombophilic condition in the United States. The fibrin clot is cleaved (fibrinolysis) by the protein plasmin, a serine protease that is generated from plasminogen by plasminogen activators such as tissue plasminogen activator (TPA, t-PA).


Biochemistry_Lippinco

Recombinant TPA is used clinically.

Biochemistry_Lippinco. Recombinant TPA is used clinically.


Biochemistry_Lippinco

Biochemistry_Lippinco. Wound to a tissue damages blood vessels and exposes collagen. Platelets (thrombocytes) adhere to the exposed collagen, get activated, and aggregate to form a platelet plug. Adhesion is mediated by von Willebrand factor (VWF). VWF binds collagen, and platelets bind VWF via glycoprotein Ib (GPIb) within a receptor complex on the platelet surface. Deficiency of VWF results in von Willebrand disease, the most common inherited coagulopathy. Once adhered, platelets get activated. Platelet activation involves changes in shape (discoidal to spherical with pseudopodia) and degranulation, the process by which platelets release the contents of their storage granules. Thrombin is the most potent activator of platelets. Thrombin binds to protease-activated G protein–coupled receptors on the surface of platelets. Activated platelets release substances that cause vasoconstriction (serotonin and thromboxane A2 [TXA2]), recruit and activate other platelets (adenosine diphosphate and TXA2), and support


Biochemistry_Lippinco

Biochemistry_Lippinco. platelets. Activated platelets release substances that cause vasoconstriction (serotonin and thromboxane A2 [TXA2]), recruit and activate other platelets (adenosine diphosphate and TXA2), and support the formation of a fibrin clot (FV, FXIII, and fibrinogen). Activation causes changes in platelets that lead to their aggregation. Structural changes in a surface receptor (GPIIb/IIIa) expose binding sites for fibrinogen. Fibrinogen molecules link activated platelets to one another. The fibrinogen is activated to fibrin by thrombin and then cross-linked by FXIIIa coming both from the blood and from platelets. The initial loose plug of platelets (primary hemostasis) is strengthened by the fibrin meshwork (secondary hemostasis). Disorders of platelets and coagulation proteins can result in deviations in the ability to clot. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are clinical laboratory tests used to evaluate the clotting cascade.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. For Questions 31.1–31.5, match the most appropriate protein factors (F) of clotting to the description. 5.1. This factor activates components of the intrinsic, extrinsic, and common pathways. 5.2. This factor converts the soluble clot to an insoluble clot. 5.3. This factor initiates the common pathway. 5.4. This factor is an accessory protein that potentiates the activity of factor Xa. 5.5. This factor is a γ-carboxyglutamate–containing serine protease of the extrinsic pathway.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5.4. This factor is an accessory protein that potentiates the activity of factor Xa. 5.5. This factor is a γ-carboxyglutamate–containing serine protease of the extrinsic pathway. Correct answers = B, J, H, D, E. Thrombin (FII) is formed in the common pathway and activates components in each of the three pathways of the clotting cascade. FXIII, a transglutaminase, covalently cross-links associated fibrin monomers, thereby converting a soluble clot to an insoluble one. The generation of FXa by the intrinsic and extrinsic pathways initiates the common pathway. FV increases the activity of FXa. It is one of three accessory (nonprotease) proteins. The others are FIII (tissue factor) and FVIII (complexes with FIX to activate FX). FVII is a γ-carboxyglutamate–containing serine protease that complexes with FIII in the extrinsic pathway. 5.6. In which patient would prothrombin time be unaffected and activated partial thromboplastin time be prolonged? A. A patient on aspirin therapy


Biochemistry_Lippinco

Biochemistry_Lippinco. 5.6. In which patient would prothrombin time be unaffected and activated partial thromboplastin time be prolonged? A. A patient on aspirin therapy B. A patient with end-stage liver disease C. A patient with hemophilia D. A patient with thrombocytopenia Correct answer = C. Prothrombin time (PT) measures the activity of the extrinsic through the common pathways, and activated partial thromboplastin time (aPTT) measures the activity of the intrinsic through the common pathways. Patients with hemophilia are deficient in either FVIII (hemophilia


Biochemistry_Lippinco

Biochemistry_Lippinco. A) or FIX (hemophilia B), components of the common pathway. They have an intact extrinsic pathway. Therefore, the PT is unaffected, and the aPTT is prolonged. Patients on aspirin therapy and those with thrombocytopenia have alterations in platelet function and number, respectively, and not in the proteins of the clotting cascade. Therefore, both the PT and the aPTT are unaffected. Patients with end-stage liver disease have decreased ability to synthesize clotting proteins. They show prolonged PT and aPTT. 5.7. Which one of the following can be ruled out in a patient with thrombophilia? A. A deficiency of antithrombin III B. A deficiency of FIX C. A deficiency of protein C D. An excess of prothrombin E. Expression of FV Leiden Correct answer = B. Symptomatic deficiencies in clotting factors will present with a decreased ability to clot (coagulopathy). Thrombophilia, however, is characterized by an increased tendency to clot. Choices A, C, D, and E result in thrombophilia.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5.8. Current guidelines for the treatment of patients with acute ischemic stroke (a stroke caused by a blood clot obstructing a vessel that supplies blood to the brain) include the recommendation that tissue plasminogen activator (TPA) be used shortly after the onset of symptoms. The basis of the recommendation for TPA is that it activates: A. antithrombin III. B. the activated protein C complex. C. the receptor for von Willebrand factor. D. the serine protease that degrades fibrin. E. thrombomodulin.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. antithrombin III. B. the activated protein C complex. C. the receptor for von Willebrand factor. D. the serine protease that degrades fibrin. E. thrombomodulin. Correct answer = D. TPA converts plasminogen to plasmin. Plasmin (a serine protease) degrades the fibrin meshwork, removing the obstruction to blood flow. Antithrombin III in association with heparin binds thrombin and carries it to the liver, decreasing thrombin's availability in the blood. The activated protein C complex degrades the accessory proteins FV and FVIII. The platelet receptor for von Willebrand factor is not affected by TPA. Thrombomodulin binds thrombin and converts it from a protein of coagulation to one of anticoagulation by decreasing its activation of fibrinogen and increasing its activation of protein C.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5.9. The adhesion, activation, and aggregation of platelets provide the initial plug at the site of vessel injury. Which of the following statements concerning the formation of this platelet plug is correct? A. Activated platelets undergo a shape change that decreases their surface area. B. Formation of a platelet plug is prevented in intact vessels by the production of thromboxane A2 by endothelial cells. C. The activation phase requires production of cyclic adenosine monophosphate. D. The adhesion phase is mediated by the binding of platelets to von Willebrand factor via glycoprotein Ib. E. Thrombin activates platelets by binding to a protease-activated G protein–coupled receptor and causing activation of protein kinase A.


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Thrombin activates platelets by binding to a protease-activated G protein–coupled receptor and causing activation of protein kinase A. Correct answer = D. The adhesion phase of platelet plug formation is initiated by the binding of von Willebrand factor to a receptor (glycoprotein Ib) on the surface of platelets. Shape change from discoidal to spherical with pseudopodia increases the surface area of platelets. Thromboxane A2 is made by platelets. It causes platelet activation and vasoconstriction. Adenosine diphosphate is released from activated platelets, and it itself activates platelets. Thrombin works primarily through receptors coupled to Gq proteins causing activation of phospholipase C. 5.11. Nephrotic syndrome is a kidney disease characterized by protein loss in the urine (≥3 g/day) that is accompanied by edema. The loss of protein results in a hypercoagulable state. Excretion of which of the following proteins would explain the thrombophilia seen in the syndrome?


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Antithrombin III B. FV C. FVIII D. Prothrombin Correct answer = A. Antithrombin III (ATIII) inhibits the action of thrombin (FIIa), a Gla-containing protein of clotting that activates the extrinsic, intrinsic, and common pathways. Excretion of ATIII in nephrotic syndrome allows the actions of FIIa to continue, resulting in a hypercoagulable state. The other choices are proteins required for clotting. Their excretion in the urine would decrease clotting. 5.12. Blocking the action of which of the following proteins would be a rational therapy for hemophilia B? A. FIX B. FXIII C. Protein C D. Tissue factor pathway inhibitor


Biochemistry_Lippinco

Biochemistry_Lippinco. 5.12. Blocking the action of which of the following proteins would be a rational therapy for hemophilia B? A. FIX B. FXIII C. Protein C D. Tissue factor pathway inhibitor Correct answer = D. Hemophilia B is a coagulopathy caused by decreased thrombin production by the common pathway as a result of a deficiency in FIX of the intrinsic pathway. Because the extrinsic pathway also can result in thrombin production, blocking the inhibitor of this pathway (tissue factor pathway inhibitor) should, in principle, increase thrombin production. 5.13. The parents of a newborn baby girl refuse to allow the baby to be given the injection of vitamin K that is recommended shortly after birth to prevent vitamin K deficiency bleeding, which is caused by the low levels of the vitamin in newborns. The activity of which one of the following protein factors involved in clotting would be decreased with vitamin K deficiency? A. FV B. FVII C. FXI D. FXII E. FXIII


Biochemistry_Lippinco

Biochemistry_Lippinco. A. FV B. FVII C. FXI D. FXII E. FXIII Correct answer = B. FVII is a γ-carboxyglutamate (Gla)-containing protein of clotting. The creation of Gla residues by γ-glutamyl carboxylase requires vitamin K as a coenzyme. FII, FIX, and FX, as well as proteins C and S that limit clotting, also contain Gla residues. The other choices do not contain Gla residues. 5.14. Thrombin, produced in the common pathway of clotting, has both procoagulant and anticoagulant activities. Which one of the following is an anticoagulant activity of thrombin? A. Activating FXIII B. Binding to thrombomodulin C. Increasing nitric oxide production D. Inhibiting FV and FVIII E. Inhibiting platelet activation F. Inhibiting tissue factor pathway inhibitor


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Activating FXIII B. Binding to thrombomodulin C. Increasing nitric oxide production D. Inhibiting FV and FVIII E. Inhibiting platelet activation F. Inhibiting tissue factor pathway inhibitor Correct answer = B. Thrombin bound to thrombomodulin activates protein C that degrades the accessory proteins FV and FVIII, thereby inhibiting clotting. Activation of FXIII by thrombin strengthens the fibrin clot. Nitric oxide, a vasodilator made by endothelial cells, decreases clot formation. It is not affected by thrombin. Thrombin is a powerful activator of platelets. Inhibition of tissue factor pathway inhibitor would increase clotting. 5.15. A student is reviewing the use of prothrombin time (PT) and activated partial thromboplastin time (aPTT) in evaluating a suspected deficiency of a clotting protein. Which one of the following results would be correct for a deficiency in FXIII? A. Both prothrombin time and activated partial thromboplastin time are decreased.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Both prothrombin time and activated partial thromboplastin time are decreased. B. Both prothrombin time and activated partial thromboplastin time are increased. C. Both prothrombin time and activated partial thromboplastin time are unchanged. D. Only prothrombin time is affected. E. Only activated partial thromboplastin time is affected. Correct answer = C. FXIII is a transglutaminase that cross-links fibrin molecules in a soft clot to form a hard clot. Its deficiency does not affect the PT or aPTT tests. [Note: It is evaluated by a clot solubility test.] 5.16. Why do individuals with Scott syndrome, a rare disorder caused by mutations to scramblase in platelets, have a tendency to bleed?


Biochemistry_Lippinco

Biochemistry_Lippinco. Scramblase moves phosphatidylserine (PS) from the cytosolic leaflet to the extracellular leaflet in the plasma membrane of platelets. This disrupts the asymmetrical localization of membrane phospholipids created by ATP-dependent flippases (move PS from extracellular to cytosolic leaflet) and floppases (move phosphatidylcholine [PC] in the opposite direction). Having PS on the outer face of platelet membranes provides a site for protein clotting factors to interact and activate thrombin. If scramblase is inactive, PS is not available to these factors, and bleeding results.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5.10. Several days after having had their home treated for an infestation of rats, the parents of a 3-year-old girl become concerned that she might be ingesting the poison-containing pellets. After calling the Poison Hotline, they take her to the emergency department. Blood studies reveal a prolonged prothrombin and activated partial thromboplastin time and a decreased concentration of FII, FVII, FIX, and FX. Why might administration of vitamin K be a rational approach to the treatment of this patient? Many rodent poisons are super warfarins, drugs that have a long half-life in the body. Warfarin inhibits γ-carboxylation (production of γ-carboxyglutamate, or Gla, residues), and the clotting proteins reported as decreased are the Glacontaining proteases of the clotting cascade. [Note: Proteins C and S of anticlotting are also Gla-containing proteins.] Because warfarin functions as a vitamin K antagonist, administration of vitamin K is a rational approach to treatment.


Biochemistry_Lippinco

Biochemistry_Lippinco. I. I. Integrative Cases Metabolic pathways, initially presented in isolation, are, in fact, linked to form an interconnected network. The following four integrative case studies illustrate how a perturbation in one process can result in perturbations in other processes of the network. Case 1: Chest Pain Patient Presentation: BJ, a 35-year-old man with severe substernal chest pain of ~2 hours’ duration, is brought by ambulance to his local hospital at 5 AM. The pain is accompanied by dyspnea (shortness of breath), diaphoresis (sweating), and nausea.


Biochemistry_Lippinco

Biochemistry_Lippinco. Focused History: BJ reports episodes of exertional chest pain in the last few months, but they were less severe and of short duration. He smokes (2–3 packs per day), drinks alcohol only rarely, eats a “typical” diet, and walks with his wife most weekends. His blood pressure has been normal. Family history reveals that his father and paternal aunt died of heart disease at age 45 and 39 years, respectively. His mother and younger (age 31 years) brother are said to be in good health. Physical Examination (Pertinent Findings): BJ is pale and clammy and is in distress due to chest pain. Blood pressure and respiratory rate are elevated. Lipid deposits are noted on the periphery of his corneas (corneal arcus; see left image) and under the skin on and around his eyelids (xanthelasmas; see right image). No deposits on his tendons (xanthomas) are detected.


Biochemistry_Lippinco

Biochemistry_Lippinco. Pertinent Test Results: BJ’s electrocardiogram is consistent with a myocardial infarction (MI). Angiography reveals areas of severe stenosis (narrowing) of several coronary arteries. Initial results from the clinical laboratory include the following: H = High; L = Low. [Note: BJ had not eaten for ~8 hours prior to the blood draw.] Diagnosis: MI, the irreversible necrosis (death) of heart muscle secondary to ischemia (decreased blood supply), is caused by the occlusion (blockage) of a blood vessel most commonly by a blood clot (thrombus). BJ subsequently is determined to have heterozygous familial hypercholesterolemia (FH), also known as type IIa hyperlipidemia. Immediate Treatment: BJ is given O2, a vasodilator, pain medication, and drugs to dissolve blood clots (thrombolytics) and reduce clotting (antithrombotics).


Biochemistry_Lippinco

Biochemistry_Lippinco. Immediate Treatment: BJ is given O2, a vasodilator, pain medication, and drugs to dissolve blood clots (thrombolytics) and reduce clotting (antithrombotics). Long-Term Treatment: Lipid-lowering drugs (for example, high-potency statins, bile acid [BA] sequestrants, and niacin); daily aspirin; β-blockers; and counseling on nutrition, exercise, and smoking cessation would be part of the long-term treatment plan. Prognosis: Patients with heterozygous FH have ~50% of the normal number of functional LDL receptors and a hypercholesterolemia (two to three times normal) that puts them at high risk (>50% risk) for premature coronary heart disease (CHD). However, <5% of patients with hypercholesterolemia have FH.


Biochemistry_Lippinco

Biochemistry_Lippinco. Nutrition Nugget: Dietary recommendations for individuals with heterozygous FH include limiting saturated fats to <7% of total calories and cholesterol to <200 mg/day, substituting unsaturated fats for saturated fats, and adding soluble fiber (10–20 g/day) and plant sterols (2 g/day) for their hypocholesterolemic effects. Fiber increases BA excretion. This results in increased hepatic uptake of cholesterol-rich LDL to supply the substrate for BA synthesis. Plant sterols decrease cholesterol absorption in the intestine.


Biochemistry_Lippinco

Biochemistry_Lippinco. Genetics Gem: FH is caused by hundreds of different mutations in the gene for the LDL receptor (on chromosome 19) that affect receptor amount and/or function. FH is an autosomal-dominant disease in which homozygotes are more seriously affected than heterozygotes. Heterozygous FH has an incidence of ~1:500 in the general population. It is associated with increased risk of cardiovascular disease. Genetic screening of the first-degree relatives of BJ would identify affected individuals for treatment. Review Questions: Choose the ONE best answer. RQ1. Triacylglycerols are glycerol-based lipids. Which of the following is also a glycerol-based lipid? A. Ganglioside GM2 B. Phosphatidylcholine C. Prostaglandin PGI2 D. Sphingomyelin E. Vitamin D RQ2. Statins are of benefit to patients with hypercholesterolemia because they: A. decrease a rate-limiting and regulated step of de novo cholesterol biosynthesis by inhibiting hydroxymethylglutaryl coenzyme A (HMG CoA) reductase.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. decrease a rate-limiting and regulated step of de novo cholesterol biosynthesis by inhibiting hydroxymethylglutaryl coenzyme A (HMG CoA) reductase. B. decrease expression of the gene for the LDL receptor by preventing the movement of the sterol regulatory element–binding protein-2 (SREBP 2) in complex with SREBP cleavage–activating protein (SCAP) from the membrane of the endoplasmic reticulum to the membrane of the Golgi. C. increase the oxidation of cholesterol to CO2 + H2O. D. interfere with the absorption of bile salts in the enterohepatic circulation, thereby causing the liver to take up cholesterol from the blood for use in BA synthesis. E. reduce cholesterol by increasing steroid hormone and vitamin D synthesis. RQ3. Statins are competitive inhibitors of HMG CoA reductase. Which of the following statements about competitive inhibitors is correct? A. Competitive inhibitors are examples of irreversible inhibitors.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Competitive inhibitors are examples of irreversible inhibitors. B. Competitive inhibitors increase both the apparent Michaelis constant (Km) and the apparent maximal velocity (Vmax). C. Competitive inhibitors increase the apparent Km and have no effect on the Vmax. D. Competitive inhibitors decrease both the apparent Km and the apparent Vmax. E. Competitive inhibitors have no effect on the Km and decrease the apparent Vmax. RQ4. In an MI, a blood clot forms as a result of injury to a blood vessel that leads to production of a platelet plug and a fibrin meshwork. The clot occludes the blood vessel, preventing blood flow and, therefore, delivery of O2. Destruction of the clot (thrombolysis) restores blood flow. Which one of the following is an example of a thrombolytic agent? A. Activated protein C complex B. Antithrombin III C. Aspirin D. Factor XIII E. Heparin F. Tissue plasminogen activator G. Vitamin K H. Warfarin


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Activated protein C complex B. Antithrombin III C. Aspirin D. Factor XIII E. Heparin F. Tissue plasminogen activator G. Vitamin K H. Warfarin RQ5. Decreased tissue perfusion results in hypoxia (decreased O2 availability). Relative to normoxia, in hypoxia the: A. electron transport chain will be upregulated to provide protons for ATP synthesis. B. ratio of the oxidized form of nicotinamide adenine dinucleotide (NAD+) to the reduced form (NADH) will increase. C. pyruvate dehydrogenase complex will be active. D. process of substrate-level phosphorylation will be increased in the cytosol. E. tricarboxylic acid cycle will be upregulated to provide the reducing equivalents needed for oxidative phosphorylation to occur.


Biochemistry_Lippinco

Biochemistry_Lippinco. E. tricarboxylic acid cycle will be upregulated to provide the reducing equivalents needed for oxidative phosphorylation to occur. RQ6. Genetic screening of BJ’s first-degree relatives would be accomplished by mutation analysis via polymerase chain reaction–based amplification followed by automated sequencing of the promoter region and the 18 exons of the LDL receptor gene. This process would involve the: A. generation and use of complementary DNA (cDNA). B. initiation of DNA synthesis with dideoxynucleotides. C. isolation of genomic DNA from germ cells. D. use of fluorescently labeled nucleotides. TQ1. Relative to an individual with familial defective LDL receptors, what would be the expected phenotype in an individual with familial defective apolipoprotein B-100? With apolipoprotein E-2, the isoform that only poorly binds its receptor? TQ2. Why was aspirin prescribed? Hint: What pathway of lipid metabolism is affected by aspirin?


Biochemistry_Lippinco

Biochemistry_Lippinco. TQ2. Why was aspirin prescribed? Hint: What pathway of lipid metabolism is affected by aspirin? TQ3. Heart muscle normally uses aerobic metabolism to meet its energy needs. However, in hypoxia, anaerobic glycolysis is increased. What allosteric activator of glycolysis is responsible for this effect? With hypoxia, what will be the end product of glycolysis? TQ4. One of the reasons for encouraging smoking cessation and exercise for BJ is that these changes raise the level of HDL, and elevated HDL reduces the risk for CHD. How does a rise in HDL reduce the risk for CHD? Case 2: Severe Fasting Hypoglycemia Patient Presentation: JS is a 4-month-old boy whose mother is concerned about the “twitching” movements he makes just before feedings. She tells the pediatrician that the movements started ~1 week ago, are most apparent in the morning, and disappear shortly after eating.


Biochemistry_Lippinco

Biochemistry_Lippinco. Focused History: JS is the product of a normal pregnancy and delivery. He appeared normal at birth. On his growth charts, he has been at the 30th percentile for both weight and length since birth. His immunizations are up to date. JS last ate a few hours ago. Physical Examination (Pertinent Findings): JS appears sleepy and feels clammy to the touch. His respiratory rate is elevated. His temperature is normal. JS has a protuberant, firm abdomen that appears to be nontender. His liver is palpable 4 cm below the right costal margin and is smooth. His kidneys are enlarged and symmetrical. Pertinent Test Results: H = High; L = Low.


Biochemistry_Lippinco

Biochemistry_Lippinco. Pertinent Test Results: H = High; L = Low. JS is sent to the regional children’s hospital for further evaluation. Ultrasound studies confirm hepatomegaly and renomegaly and show no evidence of tumors. A liver biopsy is performed. The hepatocytes are distended. Staining reveals large amounts of lipid (primarily triacylglycerol) and carbohydrate. Liver glycogen is elevated in amount and normal in structure. Enzyme assay using liver homogenate treated with detergent reveals <10% of the normal activity of glucose 6-phosphatase, an enzyme of the endoplasmic reticular (ER) membrane in the liver and the kidneys. Diagnosis: JS has glucose 6-phosphatase deficiency (glycogen storage disease [GSD] type Ia, von Gierke disease).


Biochemistry_Lippinco

Biochemistry_Lippinco. Diagnosis: JS has glucose 6-phosphatase deficiency (glycogen storage disease [GSD] type Ia, von Gierke disease). Treatment (Immediate): JS was given glucose intravenously, and his blood glucose level rose into the normal range. However, as the day progressed, it fell to well below normal. Administration of glucagon had no effect on blood glucose levels but increased blood lactate. JS’s blood glucose levels were able to be maintained by constant infusion of glucose. Prognosis: Individuals with glucose 6-phosphatase deficiency develop hepatic adenomas starting in the second decade of life and are at increased risk for hepatic carcinoma. Kidney glomerular function is impaired and can result in kidney failure. Patients are at increased risk for developing gout, but this rarely occurs before puberty.


Biochemistry_Lippinco

Biochemistry_Lippinco. Nutrition Nugget: Long-term medical nutrition therapy for JS is designed to maintain his blood glucose levels in the normal range. Frequent (every 2–3 hours) daytime feedings rich in carbohydrate (provided by uncooked cornstarch that is slowly hydrolyzed) and nighttime nasogastric infusion (pump assisted) of glucose are advised. Avoidance of fructose and galactose is recommended because they are metabolized to glycolytic intermediates and lactate, which can exacerbate the metabolic problems. Calcium and vitamin D supplements are prescribed.


Biochemistry_Lippinco

Biochemistry_Lippinco. Genetics Gem: GSD Ia is an autosomal-recessive disorder caused by >100 known mutations to the gene for glucose 6-phosphatase located on chromosome 17. It has an incidence of 1:100,000 and accounts for ~25% of all cases of GSD in the United States. It is one of the few genetic causes of hypoglycemia in newborns. GSD Ia is not routinely screened for in newborns. [Note: Deficiency of the translocase that moves glucose 6phosphate into the ER is the cause of GSD Ib. Hypoglycemia and neutropenia are seen.] Review Questions: Choose the ONE best answer. RQ1. JS is hypoglycemic because: A. free (nonphosphorylated) glucose cannot be produced from either glycogenolysis or gluconeogenesis as a result of the deficiency in glucose 6-phosphatase. B. glycogen phosphorylase is dephosphorylated and inactive, and glycogen cannot be degraded. C. hormone-sensitive lipase is dephosphorylated and inactive, and fatty acid substrates for gluconeogenesis cannot be generated.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. hormone-sensitive lipase is dephosphorylated and inactive, and fatty acid substrates for gluconeogenesis cannot be generated. D. the decrease in the insulin/glucagon ratio upregulates glucose transporters in the liver and kidneys, resulting in increased uptake of blood glucose. RQ2. JS was prescribed calcium supplements because chronic acidosis can cause bone demineralization, resulting in osteopenia. Vitamin D (1,25-diOH-D3) was also prescribed because vitamin D: A. binds Gq protein–coupled membrane receptors and causes a rise in inositol trisphosphate with release of calcium from intracellular stores. B. cannot be synthesized by humans and, therefore, must be supplied in the diet. C. is a fat-soluble vitamin that increases intestinal absorption of calcium. D. is the coenzyme-prosthetic group for calbindin, a calcium transporter in the intestine.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. is a fat-soluble vitamin that increases intestinal absorption of calcium. D. is the coenzyme-prosthetic group for calbindin, a calcium transporter in the intestine. RQ3. The hepatomegaly and renomegaly seen in JS are primarily the result of an increase in the amount of glycogen stored in these organs. What is the basis for glycogen accumulation in these organs? A. Glycolysis is downregulated, which pushes glucose to glycogenesis. B. Increased oxidation of fatty acids spares glucose for glycogenesis. C. Glucose 6-phosphate is an allosteric activator of glycogen synthase b. D. The rise in the insulin/glucagon ratio favors glycogenesis. RQ4. Glucose 6-phosphatase is an integral protein of the ER membrane. Which of the following statements about such proteins is correct? A. If glycosylated, the carbohydrate is on the portion of the protein that extends into the cytosol. B. They are synthesized on ribosomes that are free in the cytosol.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. If glycosylated, the carbohydrate is on the portion of the protein that extends into the cytosol. B. They are synthesized on ribosomes that are free in the cytosol. C. The membrane-spanning domain consists of hydrophilic amino acids. D. The initial targeting signal is an amino terminal hydrophobic signal sequence. TQ1. What is the likely reason for JS’s twitching movements? TQ2. Why was the liver homogenate treated with detergent? Hint: Think about where the enzyme is located. TQ3. Why is JS’s blood glucose level unaffected by glucagon? Hint: What is the role of glucagon in normal individuals who experience a drop in blood glucose? TQ4. Why are urate and lactate elevated in a disorder of glycogen metabolism? Hint: It is the result of a decrease in inorganic phosphate (Pi), but why is Pi decreased? TQ5. A.Why are triacylglycerols and cholesterol elevated? Hint: Glucose is the primary carbon source for their synthesis. B.Why are ketone bodies not elevated?


Biochemistry_Lippinco

Biochemistry_Lippinco. TQ5. A.Why are triacylglycerols and cholesterol elevated? Hint: Glucose is the primary carbon source for their synthesis. B.Why are ketone bodies not elevated? Case 3: Hyperglycemia and Hyperketonemia Patient Presentation: MW, a 40-year-old woman, was brought to the hospital in a disoriented, confused state by her husband. Focused History: As noted on her medical alert bracelet, MW has had type 1 diabetes (T1D) for the last 24 years. Her husband reports that this is her first medical emergency in 2 years. Physical Examination (Pertinent Findings): MW displayed signs of dehydration (such as dry mucous membranes and skin, poor skin turgor, and low blood pressure) and acidosis (such as deep, rapid breathing [Kussmaul respiration]). Her breath had a faintly fruity odor. Her temperature was normal.


Biochemistry_Lippinco

Biochemistry_Lippinco. Pertinent Test Results: Rapid, bedside tests were strongly positive for glucose and acetoacetate and negative for protein. Results on blood tests performed by the clinical laboratory are shown below: H=High;L=Low. Microscopic examination of her urine revealed a urinary tract infection (UTI). Diagnosis: MW is in diabetic ketoacidosis (DKA) that was precipitated by a UTI. [Note: Diabetes increases the risk for infections such as UTI.] Immediate Treatment: MW was rehydrated with normal saline given intravenously (IV). She also was given insulin IV. Blood glucose, ketone bodies, and electrolytes were measured periodically. Antibiotic treatment of her UTI was started. Long-Term Treatment: Diabetes increases the risk for macrovascular complications (such as coronary artery disease and stroke) and microvascular complications (such as retinopathy, nephropathy, and neuropathy). Ongoing monitoring for these complications will be continued.


Biochemistry_Lippinco

Biochemistry_Lippinco. Prognosis: Diabetes is the seventh leading cause of death by disease in the United States. Individuals with diabetes have a reduced life expectancy relative to those without diabetes. Nutrition Nugget: Monitoring total intake of carbohydrates is primary in blood glucose control. Carbohydrates should come from whole grains, vegetables, legumes, and fruits. Low-fat dairy products and nuts and fish rich in ω-3 fatty acids are encouraged. Intake of saturated and trans fats should be minimized. Genetics Gem: Autoimmune destruction of pancreatic β cells is characteristic of T1D. Of the genetic loci that confer risk for T1D, the human-leukocyte antigen (HLA) region on chromosome 6 has the strongest association. The majority of genes in the HLA region are involved in the immune response. Review Questions: Choose the ONE best answer. RQ1. Which of the following statements concerning T1D is correct?


Biochemistry_Lippinco

Biochemistry_Lippinco. Review Questions: Choose the ONE best answer. RQ1. Which of the following statements concerning T1D is correct? A. Diagnosis can be made by measuring the level of glucose or glycated hemoglobin (HbA1c) in the blood. B. During periods of physiologic stress, the urine of an individual with T1D would likely test negative for reducing sugars. C. T1D is associated with obesity and a sedentary lifestyle. D. The characteristic metabolic abnormalities seen in T1D result from insensitivity to both insulin and glucagon. E. Treatment with exogenous insulin allows normalization of blood glucose (euglycemia). RQ2. DKA occurs when the rate of ketone body production is greater than the rate of utilization. Which of the following statements concerning ketone body metabolism is correct? Ketone bodies: A. are made in mitochondria from acetyl coenzyme A (CoA) primarily produced by the oxidation of glucose. B. are utilized by many tissues, particularly the liver, after conversion to acetyl CoA.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. are made in mitochondria from acetyl coenzyme A (CoA) primarily produced by the oxidation of glucose. B. are utilized by many tissues, particularly the liver, after conversion to acetyl CoA. C. include acetoacetate, which can impart a fruity odor to the breath. D. require albumin for transport through the blood. E. utilized in energy metabolism are organic acids that can add to the proton load of the body. RQ3. Adipose lipolysis followed by β-oxidation of the fatty acid (FA) products is required for the generation of ketone bodies. Which of the following statements concerning the generation and use of FA is correct? A. Mitochondrial β-oxidation of FA is inhibited by malonyl CoA. B. Production of FA from adipose lipolysis is upregulated by insulin. C. The acetyl CoA product of FA β-oxidation favors the use of pyruvate for gluconeogenesis by activating the pyruvate dehydrogenase complex. D. The β-oxidation of FA utilizes reducing equivalents generated by gluconeogenesis.


Biochemistry_Lippinco

Biochemistry_Lippinco. D. The β-oxidation of FA utilizes reducing equivalents generated by gluconeogenesis. E. The FA produced by lipolysis are taken up by the brain and oxidized for energy. TQ1. At admission, MW was hypoinsulinemic, and she was given insulin. Why did MW’s hypoinsulinemia result in hyperglycemia? Hint: What is the role of insulin in glucose metabolism? TQ2. Why is there glucose in MW’s urine (glucosuria)? How is the glucosuria related to her dehydrated state? TQ3. Why is the majority of the acetyl CoA from FA β-oxidation being used for ketogenesis rather than being oxidized in the tricarboxylic acid cycle? TQ4. Was MW in positive or negative nitrogen balance when she was brought to the hospital? TQ5. What response to the DKA is apparent in MW? What response is likely occurring in the kidney? Hint: In addition to conversion to urea, how is toxic ammonia removed from the body?


Biochemistry_Lippinco

Biochemistry_Lippinco. TQ5. What response to the DKA is apparent in MW? What response is likely occurring in the kidney? Hint: In addition to conversion to urea, how is toxic ammonia removed from the body? TQ6. What would be true about the levels of ketone bodies and glucose during periods of physiologic stress in individuals with impaired FA oxidation? Case 4: Hypoglycemia, Hyperketonemia, and Liver Dysfunction Patient Presentation: AK, a 59-year-old male with slurred speech, ataxia (loss of skeletal muscle coordination), and abdominal pain, was dropped off at the Emergency Department (ED). Focused History: AK is known to the ED staff from previous visits. He has a 6year history of chronic, excessive alcohol consumption. He is not known to take illicit drugs. At this ED visit, AK reports that he has been drinking heavily in the past day or so. He cannot recall having eaten anything in that time. There is evidence of recent vomiting, but no blood is apparent.


Biochemistry_Lippinco

Biochemistry_Lippinco. Physical Examination (Pertinent Findings): The physical examination was remarkable for AK’s emaciated appearance. (His body mass index was later determined to be 17.5, which put him in the underweight category.) His facial cheeks were erythematous (red in color) due to dilated blood vessels in the skin (telangiectasia). Eye movement was normal. Neither icterus (jaundice) nor edema (swelling due to fluid retention) was seen. The liver was slightly enlarged. Bedside tests revealed hypoglycemia and hyperketonemia (as acetoacetate). Blood was drawn and sent to the clinical laboratory. Pertinent Test Results: DUI = driving under the influence; H = High; L = Low. Additional Tests: Complete blood count (CBC) and blood smear revealed a macrocytic anemia (see right image). Folate and B12 levels were ordered. Diagnosis: AK is diagnosed with alcoholism. Treatment (Immediate): Thiamine and glucose were given intravenously.


Biochemistry_Lippinco

Biochemistry_Lippinco. Diagnosis: AK is diagnosed with alcoholism. Treatment (Immediate): Thiamine and glucose were given intravenously. Prognosis: Alcoholism (alcohol dependence) is the third most common cause of preventable death in the United States. People with alcoholism are at increased risk for liver cirrhosis, pancreatitis, gastrointestinal bleeding, and some cancers. Nutrition Nugget: Those with alcoholism are at risk for vitamin deficiencies as a result of decreased intake and absorption. Thiamine (vitamin B1) deficiency is common and can have serious consequences such as Wernicke-Korsakoff syndrome with its neurologic effects. Thiamine pyrophosphate (TPP), the coenzyme form, is required for the dehydrogenase-mediated oxidation of α-keto acids (such as pyruvate) as well as the transfer of two-carbon ketol groups by transketolase in the reversible sugar interconversions in the pentose phosphate pathway.


Biochemistry_Lippinco

Biochemistry_Lippinco. Genetics Gem: Acetaldehyde, the product of ethanol oxidation by the hepatic, cytosolic, nicotinamide adenine dinucleotide (NAD+)-requiring enzyme alcohol dehydrogenase (ADH), is oxidized to acetate by the mitochondrial, NAD+-requiring aldehyde dehydrogenase (ALDH2). The majority of individuals of East Asian (but not European or African) heritage have a single nucleotide polymorphism (SNP) that renders ALDH2 essentially inactive. This results in aldehyde-induced facial flushing and mild to moderate intoxication after consumption of small amounts of ethanol. Review Questions: Choose the ONE best answer. RQ1. Many of the metabolic consequences of chronic excessive alcohol consumption seen in AK are the result of an increase in the ratio of reduced nicotinamide adenine dinucleotide (NADH) to its oxidized form (NAD+) in both the cytoplasm and mitochondria. Which of the following statements concerning the effects of the rise in mitochondrial NADH is correct?


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Fatty acid oxidation is increased. B. Gluconeogenesis is increased. C. Lipolysis is inhibited. D. The tricarboxylic acid cycle is inhibited. E. The reduction of malate to oxaloacetate in the malate–aspartate shuttle is increased. RQ2. Ethanol can also be oxidized by cytochrome P450 (CYP) enzymes, and CYP2E1 is an important example. CYP2E1, which is ethanol inducible, generates reactive oxygen species (ROS) in its metabolism of ethanol. Which of the following statements concerning the CYP proteins is correct? A. CYP proteins are heme-containing dioxygenases. B. CYP proteins of the inner mitochondrial membrane are involved in detoxification reactions. C. CYP proteins of the smooth endoplasmic reticular membrane are involved in the synthesis of steroid hormones, bile acids, and calcitriol. D. ROS such as hydrogen peroxide generated by CYP2E1 can be oxidized by glutathione peroxidase.


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Biochemistry_Lippinco. D. ROS such as hydrogen peroxide generated by CYP2E1 can be oxidized by glutathione peroxidase. E. The pentose phosphate pathway is an important source of the nicotinamide adenine dinucleotide phosphate (NADPH) that provides the reducing equivalents needed for activity of CYP proteins and the regeneration of functional glutathione. RQ3. Alcohol is known to modulate the levels of serotonin in the central nervous system, where the monoamine functions as a neurotransmitter. Which of the following statements about serotonin is correct? Serotonin is: A. associated with anxiety and depression. B. degraded via methylation by monoamine oxidase, which also degrades the catecholamines. C. released by activated platelets. D. synthesized from tyrosine in a two-step process that utilizes a tetrahydrobiopterin-requiring hydroxylase and a pyridoxal phosphate– requiring carboxylase.


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Biochemistry_Lippinco. C. released by activated platelets. D. synthesized from tyrosine in a two-step process that utilizes a tetrahydrobiopterin-requiring hydroxylase and a pyridoxal phosphate– requiring carboxylase. RQ4. Chronic, excessive consumption of alcohol is a leading cause of acute pancreatitis, a painful inflammatory condition that results from autodigestion of the gland by premature activation of pancreatic enzymes. Which of the following statements concerning the pancreas is correct? A. Autodigestion of the pancreas would be expected to result in a decrease in pancreatic proteins in the blood. B. In individuals who progress from acute to chronic pancreatitis, with the characteristic structural changes that result in decreased pancreatic function, diabetes and steatorrhea are expected findings. C. In response to secretin, the exocrine pancreas secretes protons to lower the pH in the intestinal lumen. D. Pancreatitis may also be seen in individuals with hypercholesterolemia.


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Biochemistry_Lippinco. C. In response to secretin, the exocrine pancreas secretes protons to lower the pH in the intestinal lumen. D. Pancreatitis may also be seen in individuals with hypercholesterolemia. TQ1. A. What effect does the rise in cytosolic NADH seen with ethanol metabolism have on glycolysis? Hint: What coenzyme is required in glycolysis? B. How does this relate to the fatty liver (hepatic steatosis) commonly seen in alcohol-dependent individuals? TQ2. Why might individuals with a history of gouty attacks be advised to reduce their consumption of ethanol? TQ3. Why might prothrombin time be affected in alcohol-dependent individuals? TQ4. Folate and vitamin B12 deficiencies cause a macrocytic anemia that may be seen in those with alcoholism. Why is it advisable to measure vitamin B12 levels before supplementing with folate in an individual with macrocytic anemia? Case 1: Answers to Review Questions


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Biochemistry_Lippinco. RQ1.Answer = B. Phosphatidylcholine is a glycerol-based phospholipid derived from diacylglycerol phosphate (phosphatidic acid) and cytidine diphosphatecholine. Gangliosides are derived from ceramides, lipids with a sphingosine backbone. Prostaglandins of the 2 series (such as PGI2) are derived from the 20 carbon polyunsaturated fatty acid arachidonic acid. Sphingomyelin is a sphingophospholipid derived from ceramide. Vitamin D is derived from an intermediate in the biosynthetic pathway for the sterol cholesterol. RQ2.Answer = A. Statins inhibit hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, thereby preventing the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of HMG CoA to mevalonate and decreasing cholesterol biosynthesis (see figure below). The decrease in cholesterol content caused by statins results in movement of the sterol regulatory element–binding protein-2 (SREBP-2) in complex with SREBP cleavage– activating protein (SCAP) from the endoplasmic


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Biochemistry_Lippinco. cholesterol content caused by statins results in movement of the sterol regulatory element–binding protein-2 (SREBP-2) in complex with SREBP cleavage– activating protein (SCAP) from the endoplasmic reticular membrane to the Golgi membrane. SREBP-2 is cleaved, generating a transcription factor that moves to the nucleus and binds to the sterol regulatory element upstream of the genes for HMG CoA reductase and the low-density lipoprotein (LDL) receptor, increasing their expression. Humans are unable to degrade the steroid nucleus to CO2 +


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Biochemistry_Lippinco. H2O. Bile acid (BA) sequestrants, such as cholestyramine, prevent the absorption of bile salts by the liver, thereby increasing their excretion. The liver then takes up cholesterol via the LDL receptor and uses it to make BA, thereby reducing blood cholesterol levels. Steroid hormones are synthesized from cholesterol, and vitamin D is synthesized in skin from an intermediate (7dehydrocholesterol) in the cholesterol biosynthetic pathway. Therefore, inhibition of cholesterol synthesis would be expected to decrease their production as well.


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Biochemistry_Lippinco. RQ3.Answer = C. Competitive inhibitors bind to the same site as the substrate (S) and prevent the S from binding. This results in an increase in the apparent Km (Michaelis constant, or that S concentration that gives one half of the maximal velocity [Vmax]). However, because the inhibition can be reversed by adding additional substrate, the Vmax is unchanged (see figure at right). It is noncompetitive inhibitors that decrease the apparent Vmax and have no effect on Km.


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Biochemistry_Lippinco. RQ4.Answer = F. Tissue plasminogen activator (TPA) converts plasminogen to plasmin that degrades fibrin (fibrinolysis), thereby degrading the clot (thrombolysis). Aspirin, an inhibitor of cyclooxygenase, is an antiplatelet drug. Antithrombin III (ATIII) removes thrombin from the blood, and its action is potentiated by heparin. Activated protein C (APC) complex cleaves the accessory proteins factor (F)Va and FVIIIa. ATIII and APC are involved in anticoagulation. FXIII is a transglutaminase that cross-links the fibrin meshwork. Vitamin K is a fat-soluble vitamin required for the γ-carboxylation of FII, FVII, FIX, and FX. Warfarin prevents regeneration of the functional, reduced form of vitamin K. RQ5.Answer = D. In hypoxia, substrate-level phosphorylation in glycolysis provides ATP. Oxidative phosphorylation is inhibited by the lack of O2. Because the rate of ATP synthesis by oxidative phosphorylation controls the rate of cellular respiration, electron transport is inhibited. The


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Biochemistry_Lippinco. phosphorylation is inhibited by the lack of O2. Because the rate of ATP synthesis by oxidative phosphorylation controls the rate of cellular respiration, electron transport is inhibited. The resulting rise in the ratio of the reduced form of nicotinamide adenine dinucleotide (NADH) to the oxidized form (NAD+) inhibits the tricarboxylic acid cycle and the pyruvate dehydrogenase complex. RQ6.Answer = D. Fluorescently labeled nucleotides allow the base sequence of the DNA of interest to be determined. Complementary DNA (cDNA) is generated from processed messenger RNA and would not contain the promoter. Dideoxynucleotides lack the 3′-OH needed to form the 3′→5′-phosphodiester bond that joins the nucleotides and, thus, will terminate DNA synthesis. Genomic DNA obtained from white cells isolated from a blood sample would be the source of the DNA.


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Case 1: Answers to Thought Questions

Biochemistry_Lippinco. Case 1: Answers to Thought Questions


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Biochemistry_Lippinco. TQ1.The phenotype would be the same. In familial defective apolipoprotein (apo) B-100, LDL receptors are normal in number and function, but the ligand for the receptor is altered such that binding to the receptor is decreased. Decreased ligand–receptor binding results in increased levels of LDL in the blood with hypercholesterolemia. [Note: The phenotype would be the same in individuals with a gain-of-function mutation to PCSK9, the protease that decreases recycling of the LDL receptor, thereby increasing its degradation.] With the apo E-2 isoform, cholesterol-rich chylomicron remnants and intermediate-density lipoproteins would accumulate in blood. TQ2.Aspirin irreversibly inhibits cyclooxygenase (COX) and, therefore, the synthesis of prostaglandins (PG), such as PGI2 in vascular endothelial cells, and thromboxanes (TX), such as TXA2 in activated platelets. TXA2 promotes vasoconstriction and formation of a platelet plug, whereas PGI2 inhibits these events. Because platelets are


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Biochemistry_Lippinco. cells, and thromboxanes (TX), such as TXA2 in activated platelets. TXA2 promotes vasoconstriction and formation of a platelet plug, whereas PGI2 inhibits these events. Because platelets are anucleate, they cannot overcome this inhibition by synthesizing more COX. However, endothelial cells have a nucleus. Aspirin, then, inhibits formation of blood clots by preventing production of TXA2 for the life of the platelet. TQ3.The decrease in ATP (as the result of a decrease in O2 and, thus, a decrease in oxidative phosphorylation) causes an increase in adenosine monophosphate (AMP). AMP allosterically activates phosphofructokinase-1, the key regulated enzyme of glycolysis. The rise in glycolysis increases the production of ATP by substrate-level phosphorylation. It also increases the ratio of the reduced to oxidized forms of NAD. Under anaerobic conditions, pyruvate produced in glycolysis is reduced to lactate by lactate dehydrogenase as NADH is oxidized to NAD+. NAD+ is required for


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Biochemistry_Lippinco. of the reduced to oxidized forms of NAD. Under anaerobic conditions, pyruvate produced in glycolysis is reduced to lactate by lactate dehydrogenase as NADH is oxidized to NAD+. NAD+ is required for continued glycolysis. Because fewer ATP molecules are produced per molecule of substrate in substrate-level phosphorylation relative to oxidative phosphorylation, there is a compensatory increase in the rate of glycolysis under anaerobic conditions.


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Biochemistry_Lippinco. TQ4.High-density lipoprotein (HDL) functions in reverse cholesterol transport. It takes cholesterol from nonhepatic (peripheral) tissues (for example, the endothelial layer of arteries) and brings it to the liver (see figure on the next page). The ABCA1 transporter mediates the efflux of cholesterol to HDL. The cholesterol is esterified by extracellular lecithin–cholesterol acyltransferase (LCAT) that requires apo A-1 as a coenzyme. Some cholesteryl ester is transferred to very-low-density lipoproteins (VLDL) by cholesteryl ester transfer protein (CETP) in exchange for triacylglycerol. The remainder is taken up by a scavenger receptor (SR-B1) on the surface of hepatocytes. The liver can use the cholesterol from HDL in the synthesis of bile acids. Removal of cholesterol from endothelial cells prevents its accumulation (as cholesterol or cholesteryl ester), decreasing the risk of heart disease. [Note: In contrast, LDL carries cholesterol from the liver to peripheral tissues or back to


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Biochemistry_Lippinco. prevents its accumulation (as cholesterol or cholesteryl ester), decreasing the risk of heart disease. [Note: In contrast, LDL carries cholesterol from the liver to peripheral tissues or back to the liver.]


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Biochemistry_Lippinco. Case 2: Answers to Review Questions RQ1.Answer = A. Deficiency of glucose 6-phosphatase prevents the glucose 6phosphate generated by glycogenolysis and gluconeogenesis from being dephosphorylated and released into the blood (see figure below). Blood glucose levels fall, and a severe, fasting hypoglycemia results. [Note: JS’s symptoms appeared only recently because, at age 4 months, his feedings are less frequent.] Hypoglycemia stimulates release of glucagon, which leads to phosphorylation and activation of glycogen phosphorylase kinase that phosphorylates and activates glycogen phosphorylase. Epinephrine is also released and leads to phosphorylation and activation of hormone-sensitive lipase. However, typical fatty acids (FA) cannot serve as substrates for gluconeogenesis. The glucose transporters in the liver and kidneys are insulin insensitive.


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Biochemistry_Lippinco. RQ2.Answer = C. Vitamin D is a fat-soluble vitamin that functions as a steroid hormone. In complex with its intracellular nuclear receptor, it increases transcription of the gene for calbindin, a calcium (Ca2+) transporter protein in the intestine (see figure at right). Vitamin D does not bind to a membrane receptor and does not produce second messengers. It can be synthesized in the skin by the action of ultraviolet light on an intermediate of cholesterol synthesis, 7dehydrocholesterol. Of the fat-soluble vitamins (A, D, E, and K), only K functions as a coenzyme.


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Biochemistry_Lippinco. RQ3.Answer = C. Glucose 6-phosphate is a positive allosteric effector of the covalently inhibited (phosphorylated) glycogen synthase b. With the rise in glucose 6-phosphate, glycogen synthesis is activated, and glycogen stores are increased in both the liver and the kidneys. The increased availability of glucose 6-phosphate also drives glycolysis. The increase in glycolysis provides substrates for lipogenesis, thereby increasing synthesis of FA and triacylglycerols (TAG). In hypoglycemia, the insulin/glucagon ratio is low, not high.


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Biochemistry_Lippinco. RQ4.Answer = D. Membrane proteins are initially targeted to the endoplasmic reticulum (ER) by an amino terminal hydrophobic signal sequence. Glycosylation is the most common posttranslational modification found in proteins. The glycosylated portion of membrane proteins is found on the extracellular face of the membrane. The membrane-spanning domain consists of ~22 hydrophobic amino acids. Proteins destined for secretion or for membranes, the ER lumen, Golgi, or lysosomes are synthesized on ribosomes associated with the ER. Case 2: Answers to Thought Questions


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Biochemistry_Lippinco. TQ1.The twitching is the result of the adrenergic response to hypoglycemia and is mediated by the rise in epinephrine. The adrenergic response includes tremor and sweating. Neuroglycopenia (impaired delivery of glucose to the brain) results in impairment of brain function that can lead to seizures, coma, and death. Neuroglycopenic symptoms develop if the hyperglycemia persists. TQ2.Detergents are amphipathic molecules (that is, they have both hydrophilic [polar] and hydrophobic [nonpolar] regions). Detergents solubilize membranes, thereby disrupting membrane structure. If the problem were the translocase needed to move the glucose 6-phosphate substrate into the ER, rather than the phosphatase, disruption of the ER membrane would allow the substrate access to the phosphatase. TQ3.Glucagon, a peptide hormone released from pancreatic α cells in hypoglycemia, binds its plasma membrane G protein–coupled receptor on hepatocytes. The αs subunit of the associated trimeric G protein is


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Biochemistry_Lippinco. a peptide hormone released from pancreatic α cells in hypoglycemia, binds its plasma membrane G protein–coupled receptor on hepatocytes. The αs subunit of the associated trimeric G protein is activated (guanosine diphosphate is replaced by guanosine triphosphate), separates from the β and γ subunits, and activates adenylyl cyclase that generates cyclic adenosine monophosphate (cAMP) from ATP. cAMP activates protein kinase A (PKA) that phosphorylates and activates glycogen phosphorylase kinase, which phosphorylates and activates glycogen phosphorylase. The phosphorylase degrades glycogen, generating glucose 1-phosphate that is converted to glucose 6-phosphate. With glucose 6-phosphatase deficiency, the degradative process stops here (see figure below). Consequently, administration of glucagon is unable to cause a rise in blood glucose. [Note: Epinephrine would be similarly ineffective.]


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Biochemistry_Lippinco. TQ4.The availability of inorganic phosphate (Pi) is decreased because it is trapped as phosphorylated glycolytic intermediates as a result of the upregulation of glycolysis by the rise in glucose 6-phosphate. Urate is elevated because the trapping of Pi decreases the ability to phosphorylate adenosine diphosphate (ADP) to ATP, and the fall in ATP causes a rise in adenosine monophosphate (AMP). The AMP is degraded to urate. Additionally, the availability of glucose 6-phosphate drives the pentose phosphate pathway, resulting in a rise in ribose 5-phosphate (from ribulose 5-phosphate) and, consequently, a rise in purine synthesis. Nicotinamide adenine dinucleotide phosphate (NADPH) also rises. Purines made beyond need are degraded to urate (see figure on the next page). [Note: The decrease in Pi reduces the activity of glycogen phosphorylase, resulting in increased storage of glycogen with a normal structure.] Lactate is elevated because the decrease in phosphorylation of ADP to ATP


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Biochemistry_Lippinco. in Pi reduces the activity of glycogen phosphorylase, resulting in increased storage of glycogen with a normal structure.] Lactate is elevated because the decrease in phosphorylation of ADP to ATP results in a decrease in cellular respiration (respiratory control) as a result of these processes being coupled. As a consequence, reduced nicotinamide adenine dinucleotide (NADH) from glycolysis cannot be oxidized by Complex I of the electron transport chain. Instead, it is oxidized by cytosolic lactate dehydrogenase with its coenzyme NADH as pyruvate is reduced to lactate. [Note: Pyruvate is increased as a result of the increase in glycolysis.] The lactate ionizes, releasing protons (H+) and leading to a metabolic acidosis (low pH caused here by increased production of acid). Respiratory compensation causes an increased respiratory rate.


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Biochemistry_Lippinco. TQ5.Increased glycolysis results in increased availability of glycerol 3phosphate for hepatic TAG synthesis. Additionally, some of the pyruvate generated in glycolysis will be oxidatively decarboxylated to acetyl coenzyme A (CoA). However, the tricarboxylic acid cycle is inhibited by the rise in NADH, and the acetyl CoA is transported to the cytosol as citrate. The rise of acetyl CoA in the cytosol results in increased fatty acid (FA) synthesis. Recall that citrate is an allosteric activator of acetyl CoA carboxylase (ACC). The malonyl product of ACC inhibits FA oxidation at the carnitine palmitoyltransferase I step. Because mitochondrial FA oxidation generates the acetyl CoA substrate for hepatic ketogenesis, ketone body levels do not rise. The FA gets esterified to the glycerol backbone, resulting in an increase in TAG that gets sent out of the liver as components of very-low-density lipoproteins (VLDL). [Note: The hypoglycemia results in release of epinephrine and the activation of


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Biochemistry_Lippinco. resulting in an increase in TAG that gets sent out of the liver as components of very-low-density lipoproteins (VLDL). [Note: The hypoglycemia results in release of epinephrine and the activation of TAG lipolysis with release of free FA into the blood. The FA are oxidized, with the excess used in hepatic TAG synthesis.] The acetyl CoA is also a substrate for cholesterol synthesis. Thus, the increase in glycolysis results in the hyperlipidemia seen in JS (see figure below).


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Biochemistry_Lippinco. Case 3: Answers to Review Questions RQ1.Correct answer = A. Diabetes is characterized by hyperglycemia. Chronic hyperglycemia can result in the nonenzymatic glycosylation (glycation) of hemoglobin (Hb), producing HbA1c. Therefore, measurement of glucose or


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Biochemistry_Lippinco. HbA1c in the blood is used to diagnose diabetes. In response to physiologic stress (for example, a urinary tract infection), secretion of counterregulatory hormones (such as the catecholamines) results in a rise in blood glucose. Glucose is a reducing sugar. It is type 2 diabetes (T2D) that is associated with obesity and a sedentary lifestyle and is caused by insensitivity to insulin (insulin resistance). T1D is caused by lack of insulin as a result of the autoimmune destruction of pancreatic β cells. Even individuals on a program of tight glycemic control do not achieve euglycemia. RQ2.Correct answer = E. The ketone bodies 3-hydroxybutyrate and acetoacetate are organic acids, and their ionization contributes to the proton load of the body. Ketone bodies are made in the mitochondria of liver cells using acetyl coenzyme A (CoA) generated primarily from the β-oxidation of fatty acids ([FA]; see figure on the next page). Because they are water soluble, they do not require a transporter.


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Biochemistry_Lippinco. cells using acetyl coenzyme A (CoA) generated primarily from the β-oxidation of fatty acids ([FA]; see figure on the next page). Because they are water soluble, they do not require a transporter. The liver cannot use them because it lacks the enzyme thiophorase, which moves CoA from succinyl CoA to acetoacetate for conversion to two molecules of acetyl CoA. It is the acetone released in the breath that can impart a fruity odor.


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Biochemistry_Lippinco. RQ3.Correct answer = A. Malonyl CoA, an intermediate of FA synthesis, inhibits FA β-oxidation through inhibition of carnitine palmitoyltransferase I. Lipolysis occurs when the insulin/counterregulatory hormone ratio decreases. Acetyl CoA, the product of FA β-oxidation, inhibits the pyruvate dehydrogenase (PDH) complex through activation of PDH kinase and activates pyruvate carboxylase. Acetyl CoA, then, pushes pyruvate to gluconeogenesis. β-Oxidation generates reduced nicotinamide adenine dinucleotide (NADH), the reducing equivalent required for gluconeogenesis. FA are not readily catabolized for energy by the brain. Case 3: Answers to Thought Questions


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Biochemistry_Lippinco. Case 3: Answers to Thought Questions TQ1.Hypoinsulinemia results in hyperglycemia because insulin is required for the uptake of blood glucose by muscle and adipose tissue. Their glucose transporter (GLUT-4) is insulin dependent in that insulin is required for movement of the transporter to the cell surface from intracellular storage sites. Insulin is also required to suppress hepatic gluconeogenesis. Insulin suppresses the release of glucagon from pancreatic α cells. The resulting rise in the insulin/glucagon ratio results in the dephosphorylation and activation of the kinase domain of bifunctional phosphofructokinase-2 (PFK-2). The fructose 2,6bisphosphate produced by PFK-2 activates phosphofructokinase-1 of glycolysis (see figure below). It also inhibits fructose 1,6-bisphosphatase (FBP-2), thereby inhibiting gluconeogenesis. With hypoinsulinemia, the failure to take up glucose from the blood while simultaneously sending it out into the blood results in hyperglycemia.


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Biochemistry_Lippinco. TQ2.The blood glucose level has exceeded the capacity of the kidney to reabsorb glucose (via a sodium-dependent glucose transporter [SGLT]). The high concentration of glucose in the urine osmotically draws water from the body. This causes increased urination (polyuria) with loss of water that results in dehydration. TQ3.The NADH generated in FA β-oxidation inhibits the tricarboxylic acid (TCA) cycle at the three NADH-producing dehydrogenase steps. This shifts acetyl CoA away from oxidation in the TCA cycle and toward use as a substrate in hepatic ketogenesis.


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Biochemistry_Lippinco. TQ4.MW was in negative nitrogen balance: More nitrogen was going out than coming in. This is reflected in the elevated blood urea nitrogen (BUN) level seen in the patient (see figure at top right). [Note: The BUN value also reflects dehydration.] Muscle proteolysis and amino acid catabolism are occurring as a result of the fall in insulin. (Recall that skeletal muscle does not express the glucagon receptor.) Amino acid catabolism produces ammonia (NH3), which is converted to urea by the hepatic urea cycle and sent into the blood. [Note: Urea in the urine is reported as urinary urea nitrogen.] TQ5.The Kussmaul respiration seen in MW is a respiratory response to the metabolic acidosis. Hyperventilation blows off CO2 and water, reducing the concentration of protons (H+) and bicarbonate (HCO3−) as reflected in the following equation: The renal response includes, in part, the excretion of H+ as ammonium (NH4+). Degradation of branched-chain amino acids in skeletal muscle results in the


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Biochemistry_Lippinco. as reflected in the following equation: The renal response includes, in part, the excretion of H+ as ammonium (NH4+). Degradation of branched-chain amino acids in skeletal muscle results in the release of large amounts of glutamine (Gln) into the blood. The kidneys take up and catabolize the Gln, generating NH3 in the process. The NH3 is converted to NH4+ by secreted H+ and is excreted (see figure at middle right). [Note: When ketone bodies are plentiful, enterocytes shift to using them as a fuel instead of


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Biochemistry_Lippinco. Gln. This increases the amount of Gln going to the kidney.] TQ6.Because FA β-oxidation supplies the acetyl CoA substrate for ketogenesis, impaired β-oxidation decreases the ability to make ketone bodies. Ketone bodies are an alternate to the use of glucose, and, thus, dependence on glucose increases. Because FA β-oxidation supplies the NADH and the nucleoside triphosphates needed for gluconeogenesis, glucose production decreases. The result is a hypoketotic hypoglycemia. Recall that this was seen with medium-chain acyl CoA dehydrogenase (MCAD) deficiency. Case 4: Answers to Review Questions


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Biochemistry_Lippinco. RQ1.Answer = D. The rise in reduced nicotinamide adenine dinucleotide (NADH) in the mitochondria decreases the tricarboxylic acid (TCA) cycle, fatty acid (FA) oxidation, and gluconeogenesis. NADH inhibits the isocitrate dehydrogenase reaction, the key regulated step of the TCA cycle, and the αketoglutarate dehydrogenase reaction (see figure at bottom right). It also favors the reduction of oxaloacetate (OAA) to malate (not malate to OAA), decreasing the availability of OAA for condensation with acetyl coenzyme A (CoA) in the TCA cycle and for gluconeogenesis. FA oxidation requires the oxidized form of nicotinamide adenine dinucleotide (NAD+) for the 3-hydroxyacyl CoA dehydrogenase step and, thus, is inhibited by the rise in NADH. The decrease in FA oxidation decreases the production of ATP and acetyl CoA (the allosteric activator of pyruvate carboxylase) needed for gluconeogenesis. Lipolysis is activated in fasting as a consequence of the fall in insulin and the rise in catecholamines


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Biochemistry_Lippinco. and acetyl CoA (the allosteric activator of pyruvate carboxylase) needed for gluconeogenesis. Lipolysis is activated in fasting as a consequence of the fall in insulin and the rise in catecholamines that result in activation of hormone-sensitive lipase.


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Biochemistry_Lippinco. RQ2.Answer = E. The irreversible, oxidative portion of the pentose phosphate pathway provides the nicotinamide adenine dinucleotide phosphate (NADPH) that supplies the reducing equivalents needed for activity of cytochrome P450 (CYP) proteins and for the regeneration of functional (reduced) glutathione. It is also an important source of NADPH for reductive biosynthetic processes in the cytosol, such as FA and cholesterol synthesis. [Note: Malic enzyme is another source.] CYP proteins are monooxygenases (mixed-function oxidases). They incorporate one O atom from O2 into the substrate as the other is reduced to water. It is the CYP proteins of the smooth endoplasmic reticular membrane that are involved in detoxification reactions. Those of the inner mitochondrial membrane are involved in the synthesis of steroid hormones, bile acids, and vitamin D. Reactive oxygen species are reduced by glutathione peroxidase as glutathione is oxidized. RQ3.Answer = C. Serotonin is released by activated


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Biochemistry_Lippinco. synthesis of steroid hormones, bile acids, and vitamin D. Reactive oxygen species are reduced by glutathione peroxidase as glutathione is oxidized. RQ3.Answer = C. Serotonin is released by activated platelets and causes vasoconstriction and platelet aggregation. [Note: Platelets do not synthesize serotonin, but they take up that which was made in the intestine and secreted into the blood.] Serotonin is associated with a feeling of well-being. It is degraded to 5-hydroxyindoleacetic acid by monoamine oxidase that catalyzes oxidative deamination. It is catechol-O-methyltransferase that catalyzes the methylation step in the degradation of the catecholamines. Serotonin is synthesized from tryptophan in a two-step process that utilizes tetrahydrobiopterin (BH4) requiring tryptophan hydroxylase and a pyridoxal phosphate (PLP)-requiring decarboxylase (see figure at right). RQ4.Answer = B. The exocrine pancreas secretes enzymes required for the digestion of dietary carbohydrate, protein, and