N.V. Bhagavan

MUTATIONS IN A SPECIFIC HUMAN SERUM ALBUMIN THYROXINE BINDING SITE DEFINE THE STRUCTURAL BASIS OF FAMILIAL DYSALBUMINEMIC HYPERTHYROXINEMIA

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. Site-directed mutagenesis and a yeast protein expression system were used to synthesize wild type HSA and FDH HSA as well as several other HSA mutants. Studies on the binding of thyroxine to these HSA species using equilibrium dialysis and quenching of tryptophan 214 fluorescence suggest that the FDH mutation affects a single thyroxine binding site located in the 2A subdomain of HSA. Site-directed mutagenesis of HSA and thyroxine analogs were used to obtain information about the mechanism of thyroxine binding to both wild type and FDH HSA. These studies suggest that the guanidino group of arginine at amino acid position 218 in wild type HSA is involved in an unfavorable binding interaction with the amino group of thyroxine, whereas histidine at amino acid position 218 in FDH HSA is involved in a favorable binding interaction with thyroxine. Neither arginine at amino acid position 222 nor tryptophan at amino acid position 214 appears to favorably influence the binding of thyroxine to wild type HSA.

Water, Electrolytes, and Acid–Base Balance

The concentrations of various electrolytes, pH, and water balance are determined by many interwoven systems to maintain homeostasis. The composition and volume of extracellular fluid are regulated by complex hormonal and nervous system mechanisms that coordinate to control osmolality, volume, and pH. The osmolality of extracellular fluid is due mainly to Na + and accompanying anions. The kidneys are the major organs that regulate extracellular fluid composition and volume via their functional units known as nephrons. Kidneys regulate acid–base balance by excreting nonvolatile acids and conserving HCO 3 – . Lungs regulate volatile acid CO 2 excretion. These functions are coordinated and any imbalance results in metabolic and respiratory acid–base disorders. The acid–base disorders are assessed by measuring arterial blood gases and pH values, venous blood electrolyte concentrations, and serum and urinary anion gap values. These disorders often occur as complex conditions. Despite considerable variation in fluid intake, an individual maintains water balance and a constant composition of body fluids. The homeostatic regulation of water is also discussed in the chapter.

Chapter 4 – Three-Dimensional Structure of Proteins

The protein structure is defined based upon the four hierarchical structures: primary, secondary, tertiary, and quaternary. The primary structure is the unique sequence of amino acids residues, determined by DNA sequence. Secondary structure consists of four structural motifs: α-helix, β-pleated sheet, β-turns, and non-repetitive structure. Tertiary structure defines the overall three-dimensional conformation, which consists of folding of the motifs to bring together disparate amino acid residues in the assembly of a functional unit. The three-dimensional structure is stabilized by the nature of the R-groups that allows for hydrophobic interactions, to achieve maximum van der Waals forces, electrostatic interactions, and H-bonds. Formation of disulfide covalent bonds between two strategically located cysteine residues stabilizes the folded conformation. Quaternary structure refers to those proteins which consist of more than one polypeptide chain and their assembly into a functional molecule. The chapter also discusses the degradation of protein, which occurs in the body by several different processes.

Protein and Amino Acid Metabolism

This chapter provides an overview of overall metabolism of proteins and amino acids. Body protein is maintained by the balance between the rates of protein synthesis and breakdown. These processes are influenced by hormones and energy supply. Proteins constantly undergo breakdown and synthesis, which is known as protein turnover which changes during different physiologic and pathologic states of life. Proper maintenance of protein turnover rates for all of its constituent 20 amino acids is required. The bodys requirements for amino acids are met by dietary sources, which provide both essential and nonessential amino acids. The chapter also discusses protein and energy malnutrition that results in severe clinical manifestations. Kwashiorkar disorder is due to inadequate intake of protein and Marasmus disorder is due to deficiency of both protein and energy intake. Measurement of serum transthyretin serves as a marker for protein malnutrition. The chapter illustrates major reactions of amino acids such as oxidative and nonoxidative reactions, which convert amino acids to their respective α-keto acids.

Metabolism of Iron and Heme

Heme, an iron-porphyrin complex, is the prosthetic group of hemoglobin, myoglobin, cytochromes, and many other proteins. Iron is required for the biosynthesis of heme and other non-heme iron containing proteins. Several specific proteins participate in the orchestration of iron metabolism. Iron metabolism consists of the absorption of dietary iron from the gastrointestinal tract, transport in the blood, storage in the liver and macrophages, and utilization in the cells requiring synthesis of iron-containing proteins. Iron deficiency causes anemia and iron excess causes iron accumulation diseases known as hemochromatosis. Ferrous iron is absorbed principally from the mature enterocytes lining the absorptive villi of the duodenum. At the apical membrane of the enterocyte, Fe 3+ is converted to Fe 2+ by ferrireductase followed by its uptake mediated by divalent transporter 1 (DMT1). The internalized Fe 2+ is either temporarily stored after conversion to Fe 3+ as ferritin, or transported across the cell for transport to the portal capillary blood circulation

Carbohydrate Metabolism I

This chapter focuses on glycolysis and the tricarboxylic acid cycle (TCA). Glucose transports to the cells are mediated by a family of transmembrane facilitative glucose transporters (GLUTs). In the gastrointestinal (GI) tract and renal tubule cells glucose is actively transported against the concentration gradient along with Na + mediated by sodium-dependent transporter (SGLT). The major glucose transporter (GLUT4) of muscle and adipose tissue cells is regulated by insulin. The chapter discusses the ten steps involved in glycolytic pathways. Three allosteric enzymes hexokinase, phosphofructokinase and pyruvate kinase mediated by positive and negative effectors regulate glycolysis. Insulin promotes glycolysis and glucagon has opposite effects. AMP activated protein kinase (AMPK) promotes glycolysis. It is noted that under anaerobic and limited oxygen supply the end product of glycolysis is lactate. In aerobic cell the end product is pyruvate, which is oxidized in the mitochondria. TCA cycle consists of series biochemical reactions in which two carbon atoms of acetyl-CoA is oxidized to CO 2 and the energy stored in the two carbon atoms is transferred to NAD + and FAD, yielding NADH and FADH 2 .

Water, Acids, Bases, and Buffers

This chapter introduces basic concepts of the properties of water, acids, bases, and buffers. Water plays a major role in all aspects of metabolism: absorption, transport, digestion, and excretion of inorganic and organic substances, as well as maintenance of body temperature. The unique properties of water are due to its structure. The reversible dissociation of water, although very weak, is important in maintaining and regulating the bodys acid-base homeostasis. An optimal acid-base balance is maintained in body fluids and cells despite large fluxes of metabolites. It is noted that a buffer system protects the body from fluctuations in pH. Metabolism produces both inorganic and organic acids. Acids generated from metabolites other than CO 2 are non-volatile and are excreted via the kidney. Non-volatile acids are lactic acid, acetoacetic acid, β-hydroxybutyrate, and acids derived from sulfur-containing amino acids and phosphorous-containing compounds. CO 2 produced by metabolism is transported as bicarbonate ion (HCO 3 - ) in the bicarbonate–carbonic system in plasma. CO 2 is eliminated in the lungs. Perturbations in this system can lead either to retention of CO 2 or to excessive loss of CO 2 .

Lipids III: Plasma Lipoproteins

This chapter discusses the structure and composition of lipoproteins. There are five major lipoproteins—namely, chylomicrons, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Chylomicrons are synthesized in the gastrointestinal tract from dietary lipids. It contains apolipoprotein B-48. It is transported to blood circulation via lymphatic channels. It is transformed to remnant chylomicrons, after the removal of the triglycerides as free fatty acids by endothelial lipoprotein lipase activated by apo CII. The chapter discusses the lipoprotein disorders. Defects in lipoprotein disorders in the vast majority of population with high total plasma cholesterol and LDL-C, and low HDL-C levels associated with CHD risk are not completely known. CHD, the major cause of morbidity and mortality are also associated with other risk factors such as diabetes, obesity, cigarette smoking, hypertension, family history of CHD, age and male sex. Thus, CHD can occur in the absence of lipoprotein abnormalities.

Endocrine Metabolism IV: Thyroid Gland

The thyroid gland consists of two lobes connected by an isthmus, positioned on the ventral surface of the trachea just below the larynx. The gland consists of spherical follicles, which are formed by a single layer of epithelial endocrine cells, which is responsible for the synthesis of thyroid hormones. The lumen of the follicle contains the storage form thyroid hormones in the form of colloidal material. Thyroid gland also contains another type of endocrine cells that are scattered around the follicles. These cells are known as parafollicular cells also called C-cells, secrete calcitonin which is involved in the calcium metabolism. The chapter discusses the synthesis and secretion of thyroid hormones mostly T 4 and small amounts of T 3 are under the regulation of cerebral cortex-hypothalamus-anterior pituitary-thyroid-peripheral tissues axis. Hypothalamus releases thyrotropin releasing hormone (TRH), stored in the median eminence, to hypothalamic-pituitary portal vein. TRH effects anterior pituitary thyrotropes to release thyroid stimulating hormone (TSH) by G-protein mediated intracellular second messenger Ca 2+ , diacylglycerol and phosphatidylinositol, pathway.

Gastrointestinal Digestion and Absorption

The chapter discusses the anatomy and physiology of the gastrointestinal (GI) tract. The GI tract consists of mouth and esophagus, stomach, small intestine, and large intestine. The GI tract is a continuous tube and consists of four concentric layers. The organs of the GI system secrete many hormones and digestive enzymes. The origin and function of major GI hormones such as gastrin, ghrelin, cholecystokinin (CCK), secretin are discussed in the chapter. Eating provokes secretion of GI hormones. Some promote plasma glucose-lowering effects through insulin secretion from the β-cells of the pancreas. These are known as incretins. Incretin mimetics are used in the treatment of type 2 diabetes. GI hormones, in addition to their digestive function, regulate satiety and hunger at the CNS level and thus play a major role in the energy homeostasis. The chapter describes digestion, absorption, and related disorders of digestion and absorption of major food substances namely Carbohydrates, Proteins, Lipids, water, and electrolytes. The chapter ends with a discussion on thermic effect of food.