Lecture: Proteoglycans and Glycoproteins

1. Describe the general structure of proteoglycansa. Except for Hyaluronic acid all other GAGs are attached to protein Proteoglycan

monomers. Each chain contains greater than 100 monosaccharides and structure resembles a bottle brush.

2. Indicate what sugar derivatives are used for GAG synthesis a. D-Glucosamine and D-Galactosamine (They are acetylated) are the sugar derivatives.

The acid derivatives are D-Glucoronic Acid and L-Iduronic Acid. (Keratan Sulfate is the exception and has D-Galactose instead of an acid sugar)

3. Discuss the general composition of GAGs and the possible bottle-brush structure of proteoglycans

a. GAGs are compressible and most are extracellular. They maintain cell shape, adhesion, migration, cell-cell communication. Made up of repeating disaccharide unit: [acidic sugar – amino sugar]n

4. Describe the synthesis of hyaluronic acid and the extracellular assembly of proteoglycan aggregates using link proteins leading to shock absorbing aggregates

a. Carbohydrate and protein are linked through a trihexoside (Gal-Gal-Xyl) and a Serine Residue. This carbohydrate-Protein complex then associates with a molecule of hyalournoic acid with the aid of small proteins called link proteins.

5. Discuss the function of hyaluronic acid related to facilitation of cell migrationa. Hyaluronic acid has anti-adhesive properties and therefore might be useful in

postsurgical wound-healing as well6. Discuss the structure and function of heparin

a. Heparin is made up of D-Glucoronic/L-Iduronic Acid and Glucosamine. It functions as an inhibitor of blood clotting. Heparin induces the release of cell surface-associated TFPI.

7. Discuss some of the roles of glycoproteins (glycocalyx, blood proteins, mucins) a. O-linked oligosaccharides determine ABO blood group determinantsb. Mucins are large proteins with negatively charges sialic acid, N-acetyl neuraminic

acid (NANA) occupy a large space, trap water and serve as protective barriers. c. Glycocalyx- cell-surface recognition (receptors) and cell surface antigenicity.

8. Outline the synthesis of O-linked glycoproteins and indicate the amino acid side chains involved

a. –OH group of Serine or Threonine linked by N-acetylgalactosamine b. Protein is synthesized in rER and then extruded into lumen of ER. Glycosylation

occurs in the golgi through action of glycosyltransferases that add sugars one at a time. Glycosylation begins with transfer of N-acetylgalactosamine to Serine or Threonine OH group. After glycosylation the glycoprotein moves through the golgi for packaging.

9. Outline the synthesis of N-linked glycoproteins and indicate the significance of dolichol-pyrophosphate and mannose involvement (see Fig.14.16 Lippincott p. 168).

a. Amide grounp of Asparagine

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b. N-linked are also synthesized in ER and Golgi. Uses a lipid dolichol and phosphorylated lipid (dolichol phosphate).

c. The protein is made in the rER and enters the lumen. Sugars are added to the dolichol pyrophosphate by membrane bound glycosyltransferases, the sugars on the dolichol pyrophosphate are then transferred onto the polypeptide chain by protein-oligosaccharide and linked to protein via an asparagine. Chains are completed in the golgi either as complex (various sugars added) or high mannose.

10. Describe the differences between the synthesis of O-linked and N-linked glycoproteinsa. O-linked adds sugars one by one to Serine and Threonine. In N-linked the sugars are

added to the Dolichol pyrophosphate and then to an Asparagine on the protein.

Lecture: Lysosomal Storage Disorders

1. Describe the general lysosomal degradation of GAGs, glycoproteins, and sphingolipidsa. The lysosomal enzymes remove one sugar unit at a time from these molecules.

2. Outline the causes of lysosomal storage disorders in general a. A specific enzyme of the degradative pathway is deficient in these disorders,

resulting in accumulation of the substrate of the pathway. The rate of biosynthesis of the compound is usually normal, but the rate of degradation is slow due to inherited deficiency of the enzyme. The substrate of the deficient enzyme accumulates in the lysosomes, resulting in swelling of the lysosomes.

3. Describe the biochemical basis of symptoms of patients with lysosomal storage disorders (hepatosplenomegaly, mental retardation, macular cherry red spot)

a. Hepatosplenomegaly- Storage of insoluble intermediates in the mononuclear phagocyte system

4. Describe the symptoms and biochemical basis of Hunter’s and Hurler’s syndrome; include the deficient enzyme and indicate the products that accumulate in the two disorders.

a. Deficiency in the breakdown of GAGs. Characterized by the accumulation of dermatan sulfate and heparan sulfate. In Hurlers syndrome deficient enzyme is Iduronidase. Urine is positive for GaGs and you have corneal clouding. Hunters syndrome is milder form and has a deficient Iduronate Sulfatase. NO corneal clouding.

5. Describe Tay-Sachs’disease and relate to the deficient enzyme, accumulating compound, (onion-shell inclusions) and the biochemical basis for the cherry-red macula

a. Deficient enzyme is β-Hexoaminidase A and the accumulating substrate is Ganglioside (GM2). Onion shell inclusions in lysosomes and Gangliosides are responsible for the cherry red spot.

6. Describe Fabry’s disease and relate to the deficient enzyme, accumulating compound, and physical appearance of patients (dark reddish skin lesions with bathing trunk distribution, impaired sweating, muscle weakness)

a. X-linked recessive disorder and the deficient enzyme is α-Galactosidase and the accumulating substrate is Globoside (ceramide trihexoside).

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7. Describe Gaucher’s disease and relate to the deficient enzyme, accumulating compound, general cell appearance (crumpled tissue paper); discuss the infantile and adult forms of Gaucher’s disease

a. Most common lysosomal storage disorder. The deficient enzyme is β-Glucosidase and the accumulating substrate is Glucosyl ceramide (Glucocerebroside). The adult form is most common and shows marked hepatosplenomegaly and osteoporosis of long bones. The crumples tissue paper appearance of the cytoplasm of Gaucher cells is caused by enlarged elongated lysosomes filled with glucocerebrosides.

8. Describe Niemann-Pick disease and relate to the deficient enzyme, accumulating compound, general cell appearance (Foamy appearing cells); discuss Type A and Type B

a. Niemann-Pick disease is a deficiency of Sphingomyelinase and the accumulating substrate is sphingomyelin. Type A is severe infantile form and Type B appears later in childhood and presents with hepatosplenomegaly. Cherry-red spot in macula on retinal examination. Deficiency of sphingomyelinase causes lipid droplet accumulation- “Foamy cell Apearance”

9. Discuss the role of the mannose-6-P marker for transport of lysosomal enzymes into lysosomes. Indicate the biochemical defect in I-Cell disease

a. Enzymes synthesized in the ER are transported to the Golgi apparatus. In the Golgi there is phosphorylation of mannose to form mannose 6-phosphate. Enzymes that have this Mannose 6-phosphate marker are transported to the lysosomes. Enzymes without the marker are secreted by the cell. I-cell disease is when the golgi doesn’t add this marker leading to an increase of lysosomal enzymes in the blood. Accumulation of GAGs and Sphingolipids in the lysosomes. Presence of intracytoplasmic inclusions in fibroblasts and these cells are called “I-cells.” Symptoms similar to Hurler but more severe and earlier age of onset.

Lectures: Oxidation of fatty acids & ketogenesis (2 lectures)

1. Describe adipose tissue lipolysis and the regulation of hormone sensitive lipase. Indicate mechanism of fatty acid transport to the tissues

a. HS Lipase is inhibited by insulin in a well fed state and stimulated by epinephrine. It cleaves TAGs to free FA in the adipose tissue. The Free FA then are transported to the Liver and Skeletal/Cardiac muscle by Albumin. In these tissues the Free FA will undergo β-Oxidation. Fatty acids are NOT used by the brain.

2. Describe fatty acid activationa. Fatty Acids are activated to fatty acyl CoA by fatty acyl CoA synthetase (Thiokinase).

This enzyme is present in the outer mitochondrial membrane. 3. Describe the carnitine shuttle mechanism.

a. Acyl CoA cannot traverse the inner mitochondrial membrane so carnitine binds to the acyl group to form acyl-carnitine that is transported across the inner mitochondrial membrane via translocase (When this happens a carnitine comes from the matrix into the intermembrane space). Then CPT II in the matrix takes off the carnitine and the Acyl CoA is used for β-oxidation. CPT I is inhibited by malonyl CoA (Formed during FA synthesis by Acetyl CoA Carboxylase (ACC))

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4. Explain the biochemical consequences of defects in transport of fatty acids (carnitine deficiency and CPT deficiency)

a. Carnitine Deficiency- Transport of long chain fatty acids into the mitochondria is impaired and β-oxidation is decreased. You get hypoglycaemia due to impaired gluconeogenesis. (Acetyl CoA is an activator of pyruvate carboxylase). Ketogenesis is decreased if liver carnitine is deficient. Myopathic carnitine deficiency affects just muscles.

b. CPT I- characterized by a hypoclycemia and predominantly affects the liverc. CPT II- Characterized by cardiomyopathy and muscle weakness (myopathic form).

Lipid deposits are found in skeletal muscle. Prolonged exercise results in myoglobinuria and elevated CK levels.

5. List the reactions of the mitochondrial β-oxidation pathway (not substrate names)a. Acyl CoA Dehydrogenase (FAD), Enoyl CoA reductase, 3-Acyl CoA Dehydrogenase

(NAD+), Thiolase (Cleaves) 6. Outline the energetics of β-oxidation

a. Palmitoyl CoA (16 C), produce 8 Acetyl CoA, 7 NADH and 7 FADH2 7. Indicate the end products of oxidation of odd chain fatty acids

a. Products are Acetyl CoA until the end where you end up with a Propionyl CoA. This is converted to Succinyl CoA to enter the TCA cycle. Converted by Propionyl CoA carboxylase and Methylmalonyl CoA mutase

8. Outline oxidation of branched chain fatty acids and indicate the biochemical defect in Refsum’s disease.

a. α-Oxidation of branched chain fatty acids takes place in the peroxisomes. Refsum disease is a disorder characterized by deficiency of the peroxisomal phytanyl CoA α-hydroxylase. Phytanate accumulates in tissues, especially the neurologic tissues. Characterized by visual defects, ataxia and skeletal manifestations. Management includes dietary restriction of branched chain FA.

9. Distinguish between medium-chain acyl-CoA dehydrogenase deficiency and Jamaican vomiting sickness based on the pathogenetic mechanism and biochemical alterations

a. MCAD- Decreased β-Oxidation of medium chain fatty acids. C6-C10 carnitines in blood. Increased flux through ω-oxidation (Dicarboxylic Acids in Urine). Hypoglycemia because of decreased utilization of fatty acids by peripheral tissues. Increased reliance on glucose as an energy source. Decreased ATp and acetyl CoA to activate gluconeogenesis. Hypoketonemia because of decreased β-oxidation in the liver and decreased substrate for ketogenesis (Acetyl CoA). Presence of CK-MB and myoglobin in Urine. Most common inherited autosomal recessive enzyme disorder.

b. Jamaican Vomiting- Unripe Ackee fruit contains hypoglycin A which inhibits MCAD 10. Discuss Zellweger’s syndrome as a disorder of peroxisomes

a. Very long chain fatty acids (22 to 26 Carbon atoms) are initially oxidized in the peroxisomes. The shortened fatty acid is transported to mitochondria for further oxidation. Zellweger syndrome is defective peroxisomal biogenesis affecting the liver and brain. Levels of C 26 fatty acids in circulation are increased in Zellweger syndrome. Hepatomegaly and neurological manifestations are associated. Fatal.

11. Describe the pathway of hepatic ketogenesis and list the ketone bodies

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a. After β-oxidation the products of Acetyl CoA under go ketogenesis in the liver. This replenishes the NAD+ needed by 3 Hydroxyacyl CoA Dehydrogenase for β-Oxidation. The Acetyl CoA is acted upon by Thiolase, HMG CoA synthase, HMG CoA lyase and 3-Hydroxybutyrate Dehydrogenase. Acetone and 3-Hydroxy Butyrate are the ketone bodies that are formed. (Process happens in liver mitochondria). Acetone is lost via the lungs giving a fruity odor.

12. Explain ketone body utilization in peripheral tissuesa. Ketone bodies are formed in the liver and used in the peripheral tissues. 3-

Hydroxybutyrate is oxidized to acetoacetate and acetoacetate is activated to acetoacetyl CoA by Thiophorase. Thiolase then cleaves to form 2 Acetyl CoA for Krebs.

13. Prepare a concept map indicating the steps involved in the generation of ketosis in starvation and uncontrolled type 1 diabetes mellitus.

a. There is increased Ketogenesis during starvation because Acetyl CoA stimulates gluconeogenesis. Also the increased NADH/NAD+ ratio results in formation of malate from oxaloacetate for gluconeogenesis. Acetyl CoA is then used for ketogenesis rather than krebs.

b. Ketoacidosis in Diabetes Mellitus- In uncontrolled diabetes mellitus there is no insulin so there is a lot of lipolysis and uncontrolled production of ketone bodies. The production is greater than the utilization so you find ketone bodies in the urine. Ketone bodies are weak acids and lose protons resulting in acidosis. You get acetone in breath causing the fruity odor and rate of respiration increases to compensate for metabolic acidosis.

14. Correlate laboratory data in ketoacidosis (laboratory data in blood and urine) to the clinical signs in the patient; include hyperventilation

Lecture: Fatty acid synthesis

1. Indicate the main sites in the body and the metabolic condition under which fatty acid de novo synthesis takes place

a. Major sites of Fatty Acid synthesis is the Liver and lactating mammary gland. Minor sites are adipose tissue and kidney. Most likely to occur in the well-fed state. This takes place in the cell cytoplasm.

2. Outline the actions of citrate lyase and the malic enzyme in the liver.a. Acetyl CoA is converted to Citrate for transport out of the mitochondria. Citrate

Lyase is in the cytosol and converts the citrate back into OAA and Acetyl CoA. Malic Enzyme is in the cytosol and converts Malate (from the OAA by product of Citrate Lyase in the cytosol) to Pyruvate which is transported into the matrix to create Acertyl CoA. Malic enzyme also forms an NADPH.

3. Explain how acetyl-CoA carboxylase synthesizes malonyl-CoA and how the reaction is regulated (short-term and long-term)

a. First committed step of FA synthesis is catalyzed by Acetyl CoA carboxylase (ACC). RLE for FA synthesis. Needs biotin and adds a CO2 to the methyl end of acetyl CoA. This reaction requires 1 ATP per malonyl-CoA formed (inhibits CPT).

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b. Short Term Regulation- Citrate activates ACC by shifting it to the polymer. Palmitoyl CoA shifts the equilibrium towards the monomer, inhibiting ACC. Glucangon, Epinephrine, and norepinephrine all shift the equilibrium towards the monomer inhibiting ACC (phosphorylating).

c. Long Term Regulation- High carbohydrate and fat-free diets lead to increased synthesis of ACC resulting in increased synthesis of FA. High-Fat diets, fasting and glucagon lead to decreased synthesis resulting in decreased synthesis of FA.

4. Outline the fatty acid synthetic pathway and indicate the role of the acyl carrier protein in the pathway

a. The growing fatty acyl-chain on ACP makes it available for the enzyme activities of the opposite part of the dimer. If the fatty acyl-groups would be released each time, then they would have to be activated to fatty acyl-CoA which needs ATP.

b. Synthesis of Palmitate (16 C) from acetyl CoA requires 14 NADPHs, 7ATPs. Fatty Acid Synthase does all seven steps of synthesis. It links malonyl CoAs together until the final product is formed. Each time 2 NADPH are used and 1 ATP for the Malonyl CoA formation.

5. Identify the sources of NADPH for fatty acid synthesisa. Pentose Phosphate Pathway, and reaction by Malic Enzyme, Malate to Pyruvate in

the cytosol. 6. Discuss malonyl-CoA inhibition of carnitine-palmitoyl transferase I (CPT I)

a. Malonyl-CoA inhibits CPT I to make sure that Fatty Acid degradation does not take place at the same time as FA synthesis.

7. Outline the elongation and desaturation of fatty acids in humans. Explain the conversion of linoleic acid to arachidonic acid and α-linolenic acid to docosahexaenoic acid

a. Elongation occurs primarily in the membranes of the ER and involves the addition of malonyl CoAs to palmitate. In the mitochondria (minor pathway) FA elongation occurs by a reversal of β-Oxidation.

b. Fatty Acid Desaturation- Sites of unsaturation are introduced by fatty acyl-CoA desaturases that introduce double bonds at positions 5,6,9. Linolenic and linoic acid have double bonds past this point and that’s why they are essential.

c. α-Linolenic Acid Docosahexaenoic Acid (PUFA). 8. List the major tissues of triacylglycerol synthesis and storage

a. Liver synthesizes TAGs and Adipose Tissue Stores them. 9. Distinguish between the glycerol-3-P pathway and the MAG pathway for triacylglycerol

synthesisa. Glycerol 3-P is formed from DHAP which is branching out of glycolysis. In the liver

it can also be formed from free glycerol by glycerol kinase. Usage of two fatty acyl-CoAs leads to phosphatidic acid which is intermediate for TAG synthesis in fat cells and liver. It is not intermediate in the MAG pathway in intestinal mucosal cells. TAGs are eventually formed from phosphatidic acid by cleavage of the phosphate leading to DAG. Usage of another fatty acyl-CoA forms TAG. (3 FA CoAs used)

b. Intestinal mucosal cells can start TAG synthesis with dietary MAG which is formed by pancreatic lipase in the lumen of the intestines. The usage of two fatty acyl-CoAs

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leads directly to TAG without phosphatidic acid as intermediate. The purpose of pancreatic lipase is to form a molecule that can be taken up into the intestinal cells and it cleaves TAG to MAG but not further on.

Lecture: Eicosanoids

1. Describe in general the formation of eicosanoids including prostaglandins, thromboxanes and leukotrienes

a. Phospholipase A2 cleaves PIP2 to generate Arachidonic Acid and Lyso-PIP2. The Arachidonic Acid is then worked on by 5-Lipoxygenase to form Leukotrienes or used by COX to form Thromboxanes and Prostaglandins. COX is rate limiting for Prostaglandin Synthesis. COX forms PGG2 which then goes on to form PGH2 which gives rise to all the thromboxanes and prostaglandins.

2. Discuss the role of cortisol in eicosanoid synthesis a. Cortisol inhibits synthesis of all eicosanoids because it doesn’t allow formation of

arachidonic acid by phospholipase A2

3. Outline the cyclic and linear pathways starting with arachidonic acid. (Look in Notebook)a. Cyclic- COX pathwayb. Linear- Leukotriene Pathway

4. Describe the general effects of PGI2 versus TXA2, and PGE2 versus PGFα2

a. PGI2- Prostacyclin and is produced by endothelium of vessels. Vasodilation, inhibits platelet aggregation and increases cAMP

b. TXA2- Thromboxane and is produced by platelets. Promotes platelet aggregation, vasoconstriction, mobilizes intracellular calcium, contraction of smooth muscle, bronchoconstriction and decrease in cAMP

c. PGE2- Vasodilation, relaxation of smooth muscled. PGF2α- Vasoconstriction, contraction of smooth muscle, bronchoconstriction.

5. Describe the two enzyme activities of prostaglandin H synthase and cyclooxygenase (COX) a. COX 1- A constitutive enzyme found in almost all tissues. Involved with normal

physiological functions of Prostacyclin and Thromboxane. Gastric protection and limiting acid secretion, maintenance of renal blood flow, vascular homeostasis and hemostasis.

b. COX 2- Non-constitutive and can be induced in a limited number of tissues in response to immune and inflammatory mediators (TNF, cytokines, tumor promoters, endotoxin). Expression of COX-2 is inhibited by glucocorticoids. Induction of COX-2 leads to increased prostaglandin synthesis which results in pain, heat, redness, swelling and fever.

c. Prostaglandin H synthase has cyclooxygenase and peroxidase activities. 6. Differentiate between COX-1 and COX-2 and discuss their inhibition by NSAIDs

a. NSAIDs, like ibuprofen and acetaminophen inhibit COX-1 and COX-2 as competitive reversible inhibitors

7. Indicate the eicosanoids formed from EPA and their biomedical activity compared to series-2 eicosanoids.

a. Eicosanoids formed from EPA (fish oil) are considered series 3. This forms PGI3 and

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TXA3. When Eicosanoids are formed from Arachidonic acid it is called the 2 series. PGI2=PGI3 in potency. However, TXA2 > TXA3 in potency. Diet rich in fish oil will favour prevention of clotting because you’ll have TXA3

8. Describe the synthesis of leukotriene LTA and the following change to LTB and to LTCa. Arachidonic acid is worked on by 5-Lipoxygenase and you get LTA. LTA is used in

mast cells to form LTC by addition of GSH. LTB is the other product of LTA and is a chemoattractant and WBC adhesion helper.

9. Indicate the synthesis of components and actions of the slow-reacting substance of anaphylaxis (SRS-A)

a. LTC is cleaved to LTD and LTE. They contain a cysteine that originated in GSH. These 3 are components of Slow-reacting substance. They are much more potent than histamine. Increase vascular permeability but severe bronchoconstriction. Vasoconstriction and lung edema

10. Discuss the biochemical basis for using corticoids, LOX-inhibitors or inhibitors of CysLT receptors in the management of asthma.

a. Corticoids would inhibit the formation of all Eicosonoids. LOX inhibitors would only affect the Leukotrienes. CysLT would affect the receptors for Leukotrienes so they would not be able to produce their effect through G-proteins. (All Eicosanoids work through G-Proteins.

Lecture: Cholesterol (27 C) metabolism

1. List the functions of cholesterol in the human bodya. Component of Cell Membranes, Precursor of bile acids and precursor of steroid

hormones and Vitamin D. 2. Outline cholesterol synthesis and discuss the branch-point at the level of farnesyl-PP

a. Synthesis requires acetyl-CoA, NADPH, and ATP. RLE is HMG CoA reductase. Occurs in the cytoplasm with enzymes in cytosol and ER. IPP (5 C) DPP (5 C) GPP (10 C) FPP (15 C) Squalene (30 C) Lanesterol (30 C) Cholesterol (27 C)

b. FPP can form dolichol, ubiquinone and protein prenylation3. Indicate the biochemical basis of Smith-Lemli-Opitz syndrome (defect in the cholesterol

biosynthetic pathway)a. A genetic defect of 7-Dehydrocholesterol Reductase. Doesn’t let you form the

double bond in ring B. Leads to microencephaly and low IQ. 4. Explain how the pathway is regulated at the level of HMG-CoA reductase

a. Cholesterol is a feedback inhibitor. Low cholesterol stimulates the release of a regulator (SREBP) protein form the ER. SREBP binds to a region in the HMG CoA reductase gene called sterol responsive element (SRE) resulting in increased transcription of the HMG CoA reductase gene. SREBP is cleaved by SCAP.

b. High intracellular concentrations of AMP stimulate AMP kinase which phosphorylates HMG-CoA and inactivates it.

c. Statin Enzymes cause upregulation (reversible inhibitors of HMG CoA reductase). This makes the cell produce more LDL receptors that take in LDL so they can get more cholesterol. This effectively limits the cholesterol in the serum.

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d. Glucagon Phosphorylates and inactivates HMG CoA reductasee. Ubiquitin- high concentration of cholesterol and mevalonate leads to degradation of

the enzyme. 5. Outline bile acid synthesis and its regulation; indicate the rate limiting step in bile acid

synthesisa. Rate limiting step in bile acid synthesis is 7-α-hydroxylase. It is inhibited by cholic

acid. Cholic acid and Chenodeoxycholic acid are the Primary bile acids. Cholesterol is hydroxylated, double bond is reduced, and is shortened by 3 carbons.

6. Describe the conjugation of primary and secondary bile salts in the liver and its significance

a. Bile acids are conjugated with glycine or taurine before they leave the liver. Action of intestinal bacteria on bile salts convert primary bile salts into secondary bile salts by removing hydroxyl groups. These prevent Cholelithiases (precipitation of Cholesterol).

7. Discuss bile composition and predict biochemical reasons for gallstone formation a. Bile is made of up of cholesterol, PC (Lecithin) and an amino acid. Deficiency of

Lecithin and/or bile salts will lead to cholesterol precipitating in the gall bladder. 8. Describe the biochemical basis for use of chenodeoxycholic acid for management of

cholelithiasisa. Chenodeoxycholic acid is the after the step of formation of cholic acid and does not

feed-back inhibit bile acid synthesis (7-α-hydroxylase). With that it can lead itself to formation of cholic acid without inhibiting anything. You will have more bile salts to increase solubility of cholesterol.

Lecture: Steroid hormones

1. Name the five classes of steroid hormones (glucocorticoids, mineralocorticoids, androgens, estrogens and progestins)

a. Cortisol (Glucocorticoid), Corticosterone and Aldosterone (Mineralocorticoids), Testosterone (Androgen), Estradiol (Estrogens) and Progesterone (Progestins)

2. Discuss steroid hormone synthesis related to STAR and desmolase (CYP11A)a. Cholesterol is used for steroid hormone synthesis and is taken up via LDL, HDL or it is

synthesized. The transport of cholesterol into the mitochondria is performed by STAR. The rate limiting step in steroid hormone synthesis is performed by desmolase and it is the cholesterol side chain cleavage enzyme (CYP 11). Needs NADPH and O2

3. Describe the steroid hormones synthesized and released from the adrenal cortex (specified for zona fasciculata, z. glomerulosa and z. reticularis)

a. Zona Fasciculata- Cortisol (Stimulated by ACTH)i. Dominant glucocorticoid found in humans. Involved in stress adaptation, and

needed with glucagon in liver for gluconeogenesis. Leads to a degradation of muscle protein. It also reduces inflammation by inhibiting Phopholipase A2. Only unbound cortisol can enter the cells. In areas of inflammation Cortisol unbinds Transcortin and enters cells stopping the release of Arachidonic Acid

b. Zona Glomerulosa- Aldosterone, Androgens (DHEA, Androstenedione)

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ii. Stimulates renal reabsorption of Na+ and excretion of K+. Aldosterone is the principal mineralocorticoid and raises blood pressure and fluid volume. It’s release is stimulated by the hormone angiotensin II formed by ACE.

c. Z. Reticularis- Release Androgens (DHEA, Androstenedione) iii. Mainly produce DHEA and adrostenedione but also some testosterone. The

weak androgens are changed in fat cells to estradiol and in peripheral tissue to testosterone.

4. Indicate the synthesis and the metabolic actions of cortisola. Synthesized from Cholesterol. The RLE is Desmolase which forms the product of

Pregnenolone and is a precursor to Cortisol. Metabolic Actions are listed above.5. Indicate the synthesis and the metabolic actions of aldosterone

a. Same as above except Pregnenolone goes to Corticosterone and then Aldosterone. 6. Indicate the synthesis of DHEA and testosterone in the adrenal cortex

a. DHEA is precursor of testosterone and LH receptor in Leydig cells increases c-AMP and PKA resulting in testosterone synthesis.

7. Describe the synthesis of testosterone in testes and of estradiol in ovariesa. Estradiol is formed from Testosterone (Testosterone is formed from DHEA).

Stimulated by FSH and controls the Menstrual cycle and promotes the development of secondary female characteristics.

8. Discuss congenital adrenal hyperplasias (CAH) and describe clinical features and biochemical basis of manifestations in patients with CYP 21 and CYP 11B and CYP 17 deficiencies

a. CYP 21/CYP 11- Cortisol cannot be formed so release of ACTh is not inhibited. ACTH stimulates adrenal cortex and more androgens are formed in Z. Reticularis as their synthesis doesn’t need CYP 21 or CYP11B. This leads to masculinzation of female fetuses/patients. CYP 21 is the most common form of CAH.

b. CYP 17- phenotypically female but can’t mature. CYP 17 is needed for DHEA synthesis and of testosterone. Deficiency leads to female phenotype. Estreogen synthesis in ovaries starts with cholesterol and needs testosterone as an intermediate. CYP 17 leads to females that are unable to mature. Aldosterone synthesis is increased in CYP 17 deficiency because there is uncontrolled stimulation of the adrenal cortex (ACTH isn’t inhibited).

c. Additional 3-β-Hydroxysteroid Dehydrogenase Deficiency- Virtually no Steroids. Patients have female like genitalia.

9. Indicate the hormone abnormality in Cushings’ and Addison’s diseasea. Cushings syndrome- High Cortisol concentration and low ACTH in plasma. Excess

protein loss and to fat distribution. (Caused by hyperfunction normally due to tumor)

b. Addison’s is high ACTH low Cortisol and aldosterone in plasma. Leads to muscle weakness, fatigue etc. (Caused by adrenal cortex atrophy due to disease, hypofunction of adrenal cortex)

10. Indicate the biochemical basis for the occurrence of hyperglycemia in Cushing’s syndromea. High Cortisol means more glucose via gluconeogenesis. Constant high levels of

cortisol leads to hyperglycemia. Cortisol also leads to the release of epinephrine

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which inhibits insulin release from the β-cells and stimulates release of glucagon from α-cells of pancreas. Cushings syndrome is characterized by increased release of glucose form the liver into the blood and by decreased uptake of glucose into muscle and fat cells due to lack of insulin (GLUT-4 will not take in glucose). More blood in plasma.

Lectures: Lipoproteins I & II (2 lectures)

1. Discuss lipid transport in the blood by lipoproteins and albumin.a. Albumin transports lipids but not cholesterol. Transports free fatty acids, bilirubin or

other lipids in its hydrophobic pockets. Lipids are non-covalently boundb. Lipoproteins are macromolecular complexes of a phospholipid monolayer with

mainly PC. Transport non-polar lipids (TAGs and CE) inside in serum. Specific proteins (apoproteins) are needed for lipoprotein functions.

2. Describe the location and function of lipoprotein lipase (heart, skeletal muscle and adipose tissue)

a. LPL is extracellular bound to the capillary walls of heart, skeletal muscle and adipose tissue. It cleaves TAGs in chylomicrons and VLDL which are TAG rich. LPL needs apo C=II for activation. The generated free fatty acids can be used for β-oxidation in the heart/muscle or stored in fat cells as TAGs or transported in blood by albumin. The generated free glycerol is taken up by the liver and can be used as glycerol backbone for TAG synthesis at insulin ruling.

b. Heart LPL has a smaller Km for TAGs. The heart needs fatty acids for energy metabolism even when plasma lipoprotein concentration is low.

c. Adipose LPL has a large Km and acts at elevated lipoprotein concentrations. It is activated by insulin [high blood glucose levels]. Fatty acids are stored as TAGs in fat cells.

3. Discuss chylomicron metabolism starting from formation to its uptake into the livera. CM release from the liver needs apo B-48. CMs join the blood circulation at the

thoracic duct. For the TAGs in CM to be cleaved the CM needs to have apo C-II (LPL needs apo C-II to cleave). CM gets both apo C-II and apo E from HDL in blood. Once the TAGs have been cleaved the big CM become smaller remnants that contain dietary CE. The apo C-II is then given back to HDL. The CM remnants are taken up by the liver and still contain the apo B-48 and apo E.

4. Discuss the transport of dietary triacylglycerols from the intestine to the peripheral tissues

a. TAGs are transported to peripheral tissues by CM and VLDL5. Describe the conversion of VLDLs into IDLs by lipoprotein lipase and the uptake of IDL by

the livera. Apo B-100 is needed for the release of VLDL from the liver. VLDL gets apo C-II and

apo E from HDLs in blood. LPL cleaves TAGs in VLDLs and the remnants of VLDLs are formed and apo C-II is given back to HDL. This VLDL remnant is called IDL. 50% of IDL is taken up into the liver via remnant receptors which recognize apo E in IDL, via LDL-receptor protein (LRP), or via LDL-receptor (apo B-100/apo E)

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b. The remaining IDLs in the blood will be used to form LDLs. Hepatic lipase (HTGL) cleaves TAGs in IDLs. Apo E is given back to HDLs and the formed LDLs have only apo B-100. 70% of LDLs are taken into the liver by LDL-receptors which recognize apo B-100. LDLs are also taken up by cells which contain LDL receptors in their plasma membrane as they are in the need for cholesterol. (receptor mediated endocytosis)

i. After a cholesterol rich meal the CM remnants lead to high free cholesterol levels in cytosol of hepatocytes reducing LDL-receptor synthesis leading to increased levels of serum LDL.

6. Describe the conversion of IDLs to LDLs performed by hepatic TAG lipase7. Discuss the uptake of LDL into the liver and extra-hepatic tissues by receptor mediated

endocytosis8. Describe the regulation of LDL-receptor synthesis

a. At high free cholesterol levels the cytosolic enxyme ACAT is activated and a reservoir of cholesteryl esters is formed. If there is still free cholesterol left in cytosol then the synthesis of HMG-CoA reductase and also synthesis of LDL receptors are down-regulated (inhibition of the activation of SREBP which would take place a low free cytosolic cholesterol levels).

9. Outline the receptor-mediated process involving LDL and LRP receptorsa. LRP receptors need both apo E and apo B-100 for uptake (IDL has both)b. LDL Receptors need apo B-100 for uptake (LDL has this because it gave it’s apo E to

HDL)10. Discuss the functions and effects of deficiency of the main apolipoproteins (apo B-48, apo

B-100, apo C-II, apo E and apo A-1)a. Apo B-48- release of CM from liver, lipoprotein receptor recognition

i. Synthesized in intestinal mucosal cellsb. Apo B-100- release of VLDL from liver, lipoprotein receptor recognition

i. Synthesized in liver cells and is largest polypeptide chain knownii. Needed for the uptake of LDL by LDL receptors of liver or extra hepatic

tissuec. Apo C-II- HDL apoprotein donation to CM or VLDL, activation of LPLd. Apo E- lipoprotein receptor recognition, HDL apoprotein donation to CM or VLDL

i. Needed for CM remnants uptake into liver by remnant receptors. Also needed for uptake of IDL by LRP.

e. Apo A-1- Activation of LCAT (PCAT)11. Describe the reverse cholesterol transport performed by HDL

a. HDLs contain apo A-1 for activation of LCAT which forms in blood CE using as substrates free cholesterol (provided by ABC transporter in plasma membrane) and PC of HDL. HDL delivers these CE to the liver. HDLs can be formed in blood by addition of phospholipids to apo A-1.

12. Describe the synthesis, location and action of lecithin: cholesterol acyltransferase (LCAT)a. This enzyme is synthesized by the liver and released into the blood where it is

activated by apo A-1. LCAT forms cholesteryl esters in blood. These CE are stored inside the HDL.

12

13. Describe biochemical basis of atherosclerosis in Tangier disease and name the defective transporter

a. Tangier disease is acquired and related to obesity, smoking, medical drugs and cholesterol reducing drugs. Very low serum HDL in childhood. It is related to a defective cholesterol ABC transporter in the plasma membrane. This leads to less substrate for LCAT and to early degradation of lipid poor apo A-1 in serum and very low HDL. Orange tonsils, and hepatosplenomegaly.

14. Describe the function of cholesterol ester transfer protein (CETP)a. CETP is the cholesteryl ester transfer protein and allows the transfer of TAGs from

VLDL into HDL in exchange for CEs from HDL into VLDL. These CE reach the liver inside of IDL or LDL.

15. Explain how cholesterol esters from HDL can reach the liver via IDL and LDLa. Once the TAGs in VLDL are cleaved by LPL they remnant is called IDL. IDL gives apo

C-II back to HDL and the remaining apo proteins apo B-100 and apo E are recognized by LRP (LDL receptor related protein) in the liver and taken up. The remaining IDL is converted to LDL by Hepatic Lipase, the LDL gives apo E to HDL. Now it only has apo B-100 which can be recognized by LDL receptors in the liver and taken up.

16. Describe the delivery of cholesterol esters to the liver by HDL2 via SRB-1 and using the phospholipase activity of hepatic lipase

a. Cholesteryl esters are taken up into the liver by scavenger receptors SR-B1 and hepatic lipase (has both lipase activity acting on TAGs in IDLs and phospholipase activity very unique). After binding to SR-B1 the phospholipid layer of HDL2 is opened by hepatic lipase and some CEs enter the liver. The phospholipid monlayer closes again and now the smaller HDL3 leaves the receptor. HDL3 can be changed to HDL2 in the blood again.

17. Explain how oxidized LDL particles are formed and how they are involved in foam cell development and atherosclerosis

a. oxLDL is formed from superoxides, nitric oxide, hydrogen peroxides oxidizing phospholipids or apo B-100. They are more easily retained by the ECM and proteoglycans and can be oxidized to ox-LDL. Ox-LDLs accumulate in macrophages via SR-A because they cannot be recognized by LDL receptors. This leads to a formation of foam cells. Plaque formation leads to atherosclerosis.

18. Discuss the significance of Lp(a) and LDL-Ba. oxLDL can be formed from LDL-pattern B which are small and dense and penetrate

more easily the endothelium.b. Lp(A) is similar to LDL but has an additional apo(a) linked to apo B-100 via a

disulphide bond which shows unusual structure of kringles. It is a structural analog to plasminogen. Apo(a) may compete with plasminogen for the binding to fibrin and reduce the removal of blood clots which could trigger MI or stroke.

19. Describe Type I, IIa, IIb, III, IV and V hyperlipidemias (causes, lipid profile abnormalities, biochemical basis of clinical manifestations) (On table)

20. Describe the biochemical basis for the use of statins and bile acid sequestering agents in hypercholesterolemia

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a. Statins inhibit as competitive inhibitors HMG-CoA reductase which is the regulated enzyme of cholesterol synthesis. When less free cholesterol is found in the liver then more LDL receptors are synthesized. More LDL receptors can take up more LDL from blood and reduce serum cholesterol

b. Bile acid sequesterants bind and trap bile acids/salts in the intestines and lead to excretion via the feces. The enterohepatic reuptake of primary and secondary bile acids is reduced and free cholesterol in the liver will be used to synthesize new bile acids. This reduces cytosolic free cholesterol levels in the liver and we find more LDL-receptor synthesis.

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Lecture: Hormonal regulation of fuel metabolism: The feed-fast cycle

1. Explain metabolic homeostasis and describe the mechanisms involved in the inter-tissue integration required for metabolic homeostasis.

a. The hormones insulin and glucagon, epinephrine and cortisol are the main hormonal regulators of blood glucose levels. CHO, lipid and protein metabolism of tissues are affected. Insulin and glucagon are hormones with opposite actions. Glucagon action is supported by epinephrine, norepinephrine, cortisol and other insulin counter hormones. Mechanisms required are Glycogenolysis, gluconeogenesis, ketogenesis and lipolysis. Regulation by substrate, serum level of hormones and nervous system.

2. Explain the special role of glucose in metabolic homeostasisa. High levels of blood glucose after a meal lead in the liver to glycolysis, glycogen

synthesis, and also synthesis of fatty acids and cholesterol. High levels of blood glucose also inhibits the release of glucagon.

3. Describe in general the pathways leading to fatty acid synthesis and cholesterol synthesis in the fed state starting with glucose.

a. Glucose is broken down into pyruvate by glycolysis. Through PDH an acetyl-CoA can be used to initiate FA synthesis and Cholesterol Synthesis. Both are stimulated by insulin.

4. Describe the roles of insulin and glucagon as the two major hormones that regulate fuel storage and mobilization.

Insulin (Dephosphorylates

via Protein Phosphotases)

Glucagon (Phosphorylates via

Protein Kinase A)Epinephrine

CHO Metabolism

Glycogen Synthesis and Glycolysis. GLUT-4 Mobilization, PPP

Glycogen degradation and

Gluconeogenesis in Liver only

Glycogen degradation in liver and muscle

Lipid Metabolism

Synthesis of FA, TAGs and cholesterol in the

liver, VLDL, TAG synthesis in fat cells

FA degradation and synthesis of ketone bodies in liver only

FA degradation and usage of ketone bodies in extra-

hepatic tissues. TAG degradation in fat

cells

Protein Metabolism

Synthesis of proteins in muscle

Usage of amino acids for

gluconeogenesis

Degradation of proteins in muscle mainly by cortisol

5. Describe the roles of epinephrine and glucocorticoids in regulation of fuel metabolisma. Epinephrine in table above.b. Glucocorticoids- Under stress situations, the pituitary gland releases ACTH which

stimulates release of cortisol from the adrenal cortex. Cortisol leads in adrenal

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medulla to methylation of norepinephrine to epinephrine and release of both catecholamines into the blood. Epinephrine inhibits insulin release from β-cells and stimulates glucagon release from α-cells of the pancreas. Cortisol induces PEP carboxykinase and favors gluconeogenesis in the liver. Cortisol leads to protein degradation in muscle. Cortisol leads to TAG degradation in certain fat cells and release of fatty acids into the blood. Epinephrine leads to glycogen degradation and TAG degradation in fat cells.

6. Describe the effect of changes in the insulin/glucagon ratio and plasma epinephrine levels on carbohydrate, lipid and protein metabolism.

a. High insulin ratio (anabolic hormone) leads to glucose uptake (GLUT-4). Glycogen synthesis, amino acid uptake, protein synthesis, usage of branched-chain amino acids for energy metabolism. Low plasma epinephrine levels.

b. Low Insulin/Glucagon ratio leads to protein degradation, amino acid release (alanine-glucose cycle) uptake of ketone bodies and fatty acids. This leads to a high plasma epinephrine ration because cortisol is stimulating the release of the catecholamines from the adrenal medulla.

7. Outline the metabolic changes that occur during the feed/fast cycle.8. Discuss pathways that are active/ inactive in each major organ/tissue during the feed/fast

cycle. Describe how these pathways are controlled and coordinated in different metabolic states. (Next Page)

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Lecture: Introduction to nitrogen metabolism

1. Explain in broad terms the concept of the ‘amino acid pool’a. The amino acid pool is defined as all the free amino acids in cells, blood and

extracellular fluid. b. Write the sources of amino acids for the amino acid pool and the utilization of

amino acids from the pooli. It is filled by dietary amino acids, filled by synthesized nonessential amino

acids or filled by amino acids generated by protein degradation. Amino acids are taken out of the pool for body protein synthesis, for synthesis of specilalized products derived by conversion of amino acids, and for degradation for energy metabolism in all cells or for synthesis of glucose/ketone bodies in hepatocytes.

c. Define and enumerate the essential and nonessential amino acids in humansi. Essential- PVT TIM HALL: Phenylalanine, Valine, Tryptophan, Threonine,

Isoleucine, Mehthionine, Histidine, Arginine, Leucine and Lysine.1. Arginine is definitely dietary essential in children2. **Deficiency of Phenylalanine and Methionine makes Tyrosine

and Cysteine essential (respectively). ii. Non-essential- Everything else not on this list **

2. Distinguish between the two major mechanisms of intracellular protein degradation:a. Proteasome and ubiquitin- Tags proteins by binding of globular non-enzymatic

protein ubiquitin (ATP Dependent). Proteasome recognized the ubiquitinated protein and cleaves the protein to peptides (ATP dependent). These cleaved peptides enter Amino Acid Pool. Degrades proteins synthesized by cells.

b. Lysosomal- Takes place in lysosomes with acid hyrdrolases. Degradation is ATP-independent. Lysosomes degrade mainly extracellular proteins, like plasma proteins or cell-surface membrane proteins. Membrane prevents degradation of cytosolic proteins allowing higher proton concentration inside lysosome.

3. Describe the role of liver and kidney in nitrogen metabolism and excretion of non-protein nitrogenous substances

a. Liver- Gets its dietary amino acids from the portal vein. The liver makes enzymes such as albumin and α/β Globulins which are crucial. Amino acids not used by liver are released into the blood amino acid pool, especially the dietary essential branched chain amino acids (valine, isoleucine, leucine). Cells release nitrogen to liver in the form of alanine or glutamine into the blood. Alanine gives one nitrogen to the liver while glutamine gives two.

b. Kidney- Kidney excretes nitrogen mostly as Urea (From Liver). Urea formed in the liver is transported to the kidneys where it is excreted in urine. Ammonia is also excreted glutamine ammonia (glutaminase drives this reaction). Uric acid from metabolism of purines. Creatinine from muscle cells.

4. Describe the mechanism of transport of amino acids in the renal tubule and in GITa. Dietary amino acids are transported against a concentration gradient by secondary

active co-transport with sodium ions into the intestinal mucosal cell. There are about

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7 different transport systems for amino acids which have overlapping specificities for different amino acids. These transporters are also found in the kidney.

5. Analyze and correlate clinical features and laboratory findings to the biochemical basis of cystinuria and Hartnup’s disease

a. Cystinuria- Tubular reabsorption of Cystine is decreased. The Cystine precipitates in the renal tubule. The COAL transporter is deficient (Cystine, Ornithine, Arginine and Lysine).

b. Hartnup’s Disease- Defect in transport of neutral amino acids like tryptophan. May lead to NAD+ (Niacin) deficiency (Pellagra). Tryptophan can be used for synthesis of niacin. Low protein diet.

6. Represent the general scheme of amino acid catabolisma. First step is the removal of nitrogen by transamination or deamination which is

followed by eventual degradation of the carbon skeleton in the TCA cycle. Transamination transfers the amino acid to an α-ketoacid and generate another amino acid which is glutamate. Transamination does not generate free ammonium ions. Deamination generates free ammonium ions and it performed in the liver and in the kidney where the ammonium ions are used for the urea cycle or are released into urine respectively.

7. Distinguish the fates of C- skeletons of amino acids: a. Glucogenic amino acids- leads to a degradation product that can be used for

gluconeogenesis. For example pyruvate or an additional molecule in the TCA cycleb. Ketogenic amino acids- Leads to acetoacetyl-CoA or acetyl-CoA. These carbons

cannot be used for gluconeogenesis but they can be used for ketone body synthesis in the liver.

c. Glucogenic & Ketogenic amino acids- Tryptophand. Enumerate examples of amino acids in the three groups

a. Glucogenic- Glutamate, Glutamine, Proline, Histidine, Arginine, Isoleucine, Valine, Methionine, Threonine, Tyrosine, Phenylalanine, Asparagine, Aspartate, Alanine, Cysteine, Glycine, Serine, Tryptophan

b. Ketogenic- Isoleucine, Phenylalanine, Tryptophan, Tyrosine and purely Ketogenic are Leucine and Lysine

8. Revise the important TCA cycle intermediates formed from the glucogenic amino acids and integrate their further fate in metabolism (Draw this Out on left side of page)

a. Link [glutamate and glutamine] to the TCA cycle and metabolismb. Link [aspartate and asparagine] to the TCA cycle and metabolismc. Link [alanine] to pyruvate and metabolism

Lecture: Disorders of Amino acid catabolism

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1. Phenylalanine and tyrosinesPhenylalanine

a. PKU I (Classic)- Phenylalanine levels in blood are elevated resulting in mousey odor of urine. You have a decreased pigmentation of skin and hair as tyrosine conversion to melanin is inhibited by the elevated Phe levels (Inhibits tyrosinase). Deficienct enzyme is Phenylalanine Hydroxylase (PAH). Need to make sure infants are on a Phe free diet.

b. PKU II (Malignant)- Deficiency of dihydrobiopterin synthesis or dihyrdrobiopterin reductase (BH2/BH4). Decreased neurotransmitter synthesis. Treatment includes dietary Phe restriction and providing dietary biopterin and precursors of the neurotransmitters. Worse prognosis than PKU I. Deficient catecholamine formation, deficient serotonin. BH4 is needed for L-Dopa and Serotonin. Sapropterin may be used in some PKU patients (synthetic form of BH4

Tyrosinemias

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a. Alkaptonuria- Inborn error of phenylalanine-tyrosine catabolism. Deficiency in Homogentisic Acid Oxidase, and homogentisic acid accumulates. Dietary restriction of Phe and Tyrosine may reduce deposition of homogentisic acid. Manifestations is darkening of urine on standing, discoloration of cartilage and CT.

b. Tyrosenemia Type I- Inborn error of phenylalanine-tyrosine catabolism. Deficiency of fumaryl acetoacetate hydrolase. Cabbage like odor of the urine. Tyrosine is needed for NT synthesis that’s why it is hard to exclude it from the diet.

2. Branched chain amino acidsMaple syrup urine disease

a. Dietary restriction of branched chain amino acids (leucine, isoleucine, valine). Presents with poor feeding, vomiting, poor weight gain. Ketosis and the characteristic odor of maple syrup in the urine. Dietary supplementation with TPP

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(vitamin B1) may be useful in those patients that have an enzyme with low coenzyme affinity.

Methylmalonic aciduria

b. Methylmalonate levels in circulation were elevated. Causes metabolic acidosis. Some children there is an improvement with vitamin B12 Cobalamin. Deficient enzyme is Methylmalonyl CoA mutase

3. Methionine and cysteineHomocystinuria

a. Outline the two possible fates of homocysteine- Defect in the metabolism of homocysteine. Deficiency of the cystathionine β-synthase. Homocysteine binds to

22

connective tissue and disrupts its structure. Some patients respond to oral vitamin B6 (PLP). Manifestations similar to Marfans. Homocysteine can also be transferred to a serine to form cysteine. This carbon skeleton can for succinyl-CoA for entry of the TCA cycle.

Lecture: Urea cycle

1. Identify the transport forms of ammonia from peripheral tissues (alanine & glutamine). Indicate the role of glutamate dehydrogenase and glutamine synthetase in glutamine formation. (P. 25 Cycle in top right Slide)

a. Glutamate Dehydrogenase can take free ammonia and α-Ketoglutarate and create Glutamate. Glutamine synthetase can take glutamate and synthesize Glutamine.

2. Describe the transamination (aminotransferase) reaction and its role in the transfer of amino groups. Indicate the importance of vitamin B6 (pyridoxal phosphate) P. 25 Top Right

3. Describe the role of glutamate dehydrogenase in donating NH3 for the urea cycle

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a. Oxidative deamination by glutamate dehydrogenase forms free ammonia. Glutamate α-Ketoglutarate + free ammonia

4. List the reactions that form ammonia in the liver:a. From Alanine: transamination requires amino acid transaminase and PLP as a

cofactor. The amino group is first transferred to α-Ketoglutarate to form glutamate. b. From Glutamate oxidative deamination by glutamate dehydrogenase forms free

ammonia. c. From Glutamine by glutaminase recction free ammonia and glutamate are formed. d. Explain the formation of ammonia in the intestine and its detoxification.

i. Bacterial ureases form ammonia in the colon. The ammonia enters the portal circulation and is delivered to the liver and a majority of the ammonia forms urea in the liver.

e. Analyze the significance of intestinal ammonia formation in patients with compromised liver function

i. Patients with Cirrhosis increases the amount of ammonia entering systemic circulation because it is not being converted to urea in the intestine. Ammonia is NOT being detoxified.

5. Regarding the urea cycle, a. Identify the sources of nitrogen for the formation of urea

i. The first N comes from Ammonia and the second N atom from aspartate1. Formation of aspartate from OAA

b. Recall the subcellular location of the urea cycle enzymes and energy utilized for the formation of urea

i. Takes place in the Liver. Two reactions in the mitochondria and three reactions in the cytosol. One urea molecule needs 4 high energy bonds.

c. Explain the regulation of the urea cycle (regulatory enzyme and mechanisms of regulation) and analyze the significance of N-acetyl glutamate

i. N-acetyl glutamate (NAG) is an absolute activator of CPS I (RLE)

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d. Compare and contrast the five inherited disorders associated with the urea cycle (Specify enzyme deficient, correlate clinical and biochemical features, and generalize the biochemical basis for management of hyperammonemia for each of the disorder)

i. All the urea cycle disorders are characterized by increased blood ammonia levels (Hyperammonemia) and blood glutamine levels. Deficiency of CPS I or OTC (first two enzymes) is most severe.

Disease Name Enzyme Deficient

Accumulating Substrate

Treatment

Hyperammonemia Type I

CPS I Ammonia

Sometimes responds to Arginine. Arginine forms NAG and high levels may stimulate

CPS I

Hyperammonemia Type II

OTCX-Linked and

most common

Carbomoyl Phosphate/Orotic

Acid

Citrullinemia ASS Citrulline Arginine may enhance citrulline excretion

Argininosuccinic Aciduria

ASL Arginino Succinate Arginine may enhance Argino Succinate excretion

Argininemia Arginase Arginine Diet of Essential Amino acids excluding Arginine

e. Low protein/high carb diet, α-Keto acid, prevention of stresses that induce catabolic state. Long term: liver transplant.

f. Indicate the biochemical basis for the use of phenylacetate, benzoic acid, α-keto acids, arginine and carbamoyl glutamate in the management of the urea cycle disorders

i. Phenylbutyrate- undergoes β-oxidation and forms phenylacetate. Phenylacetate combines with glutamine to form phenylacetylglutamine and is excreted in urine.

ii. Benzoic Acid- combines with glycine to form hippuric acid which can be excreted in the urine

6. Regarding acquired hyperammonemia:a. Identify causes of hyperammonemia

i. Liver disease due to viral or drug indeced hepatitis, alcoholic cirrhosis. In cirrhosis portal blood enters the systemic circulation without going to the liver. Ammonia in the intestine enters circulation causing neurotoxicity

b. Analyze the mechanisms of neurotoxicity of hyperammonemiai. Elevated ammonia leads to α-Ketoglutarate being converted to glutamate

via glutamate dehydrogenase enzyme (Consumption of TCA cycle

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intermediates. This leads to less ATP, reduced Na+/K+ activity. Osmotic pressure and eventual neuronal cell death.

c. Explain the biochemical basis of use of antibiotics and lactulose in the management of acquired hyperammonemia

i. Lactulose- is a disachharide that is resistant to digestion in the small intestine. Normal flora digest in the colon to produce lactic acid. Lactic acid is neutralized by NH4

+. More Nitrogen excreted in feces.ii. Antibiotics- reduction in bacterial urease in the gut.

Lecture: Conversion of amino acids to specialized products

1. Discuss the products of the amino acids listed below. For each specialized product:a. Specify the reactions and coenzymes involvedb. Analyze the biochemical significance of the specialized product

2. Phenylalanine/ tyrosine:a. Catecholamines (P. 32)

i. Biosynthetic pathways of catecholamines

ii. Explain the role of MAO and COMT during degradation (P. 33)

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iii. Describe VMA and homovanillic acid as the degradation product of the catecholamines & its clinical significance

a. VMA levels may be measured to estimate levels of epinephrine and norepinephrine produced. High levels leads to headache, sweating and tachycardia. (Pheochromocytoma)

b.b. Melanin- Albinism is where there is a complete or partial deficiency of melanin in

the skin, hair and eyes. Severe form of albinism affects the eyes (Oculocutaneous Albinism). Deficiency of the Tyrosinase enzyme so deficient conversion of tyrosine to melanin. Increased risk of skin damage on exposure to sunlight and increased risk of skin cancer. Melanin is a polymer of molecules.

3. Tryptophan:a. Serotonin- Synthesized in the gut, platelets and CNS. Synthesized from Tryptophan

i. Explain the formation of 5-HIAA formation and analyze its clinical significance

a. Serotonin is metabolized to 5-hydroxyindole acetic acid by MAO. In carcinoid syndrome a tumor of serotonin producing cells in GIT (APUD cells). This causes an increase in 5-HIAA in urine. Symptoms include diarrhea and bronchospasm.

b. Melatonini. Derived from serotonin and serotonin is derived from tryptophan.

Melatonin is a sleep inducing hormone. 4. Glutamic acid: (P 35)

a. GABA- inhibitory neurotransmitter in the central nervous system.

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5. Histidine:a. Histamine- Produced during allergic and inflammatory reactions by mast cells. It is a

vasodilator. Antihistamine drugs are used to prevent the adverse effects of allergic reactions. They do not reduce the formation of histamine they reduce the ability of histamine to function as signal to other pathways (Receptor antagonist).

6. Arginine:a. Nitric oxide- synthesized from arginine by nitric oxide synthase in endothelium of

blood vessels. Causes local vasodilation. Nitroglycerin used in treatment of myocardial ischemia is converted to NO that results in vasodilation of blood vessels and improvement of blood flow to the heart. NO has short half life and degraded rapidly

7. Creatine:a. Amino acids required for formation- Arginine, Glycine and SAM (S-Adenosyl

Methionine)b. Function of creatine-phosphate in the muscle- Accepts ~P groups from ATP when

the muscle is resting (temporary storage). Creatine donates ~P groups to ADP when muscle is contracting.

c. Excretion product of creatine (P 36)i. Explain creatinine formation- Creatine Creatinine is spontaneous

ii. Interpret the clinical significance of serum creatinine estimation- levels of ceratine kinase (CPK/CK-MB) are a good indicator of Myocardial ischemia/Myocardial damage. Serum creatinine levels are a good indicator of renal function. Serum ceratinine levels rise in acute or chronic renal failure.

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8. Glutathione:a. Amino acids required for formation- Glutamate, Cysteine and Glycineb. Revise the functions of glutathione- It is an intracellular reducing agent

(antioxidant). Important for the detoxification of toxic hydrogen peroxide especially in RBCs. It is conjugated with drugs to make them soluble, serves as a cofactor for enzymatic reactions, and serves as an aid in rearrangement of protein disulfide bonds.

9. Correlate each of the inherited/ acquired disorder listed below to the biochemical basis of the manifestations and the associated laboratory features

a. Parkinson’s disease- Parkinsons is a loss of dopamine producing cells in the basal ganglia. Neurodegenerative disorder. Characterized by movement disorders: spasticity, tremors, loss of memory, mood disturbances and postural instability. Symptoms are improved by L-DOPA. L-DOPA is converted to dopamine in the brain and that improves symptoms.

b. Pheochromocytoma- Overproduction of catecholamines. Headache, sweating, tachycardia are predominant symptoms. This can be cause by an adrenal medulla tumor, and patients will have high urinary VMA.

c. Carcinoid syndrome- In carcinoid syndrome there is a tumor of serotonin producing cells in GIT (APUD cells). This causes an increase in 5-HIAA in urine (Serotonin is metabolized to 5-HIAA by MAO). Symptoms include diarrhea and bronchospasm.

d. Albinism- Partial or complete deficiency of melanin in the skin, hair and eyes. Deficiency of the tyrosinase enzyme which converts tyrosine to melanin.

Lecture: Metabolic roles of folic acid and vitamin B12 (One carbon metabolism)

1. Specify the one- carbon donors in metabolism and the groups they donate (SAM, THF, Cobalamin)

a. SAM donates a methyl groupb. THF donates formyl, methenyl, methylene, methyl groupsc. Cobalamin donates methylcobalamin

2. Explain the formation of SAM and the reactions requiring SAMa. SAM is required for methylation (CH3) reactions. It is formed from L-Methionine by

S-Adenosyl Methionine Synthetase. SAM donates a methyl group in the synthesis of epinephrine, creatine, phospholipids like PC. After it donates it’s methyl group a homocysteine is formed.

3. Analyze the metabolism of homocysteine, vitamins/ coenzymes required for metabolism & interpret the clinical significance of homocysteine (Draw pic on Page 2 on left)

a. After SAM donates it’s methyl group it becomes homocysteine. It can then be converted back to methionine (create SAM again) or Cysteine.

b. To synthesize methionine from homocysteine the coenzymes required are vitamin B12 and Methyl THF. To synthesize cysteine from homocysteine you need PLP and Cystathione beta-synthase

c. Deficiency in either vitamin B12 or folic acid causes elevated plasma homocysteine levels. This can lead to damage of blood vessels and poses a risk for thrombosis.

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Megaloblastic anemia must be treated with both folate and vitamin B12. Other cuases of B12 deficiency are pure vegan diet, chronic pancreatitis, and terminal ileal disease (Chron’s disease). It can also lead to increased platelet aggregation, lipid oxidation and calcification of atherosclerotic plaques.

4. Explain the formation of THF from folate and specify the mechanism of action of their inhibitors

a. The precursor of THF is dietary folic acid and the required enzyme is dihydrofolate reductase in a two step reaction and requires 2 NADPH.

b. Inhibitors- methotrexate and sulphonamides. Methotrexate inhibits the human dihydrofolate reductase and sulphonamides inhibit the bacterial dihydropteroate synthetase.

5. Summarize the formation of one carbon groups from amino acid metabolism and the utilization of 1-C groups for nucleotide synthesis

a. THF can accept 1-Carbon groups from amino acids and can donate 1-Carbon groups for biosynthetic reactions such as purine and pyrimidine synthesis.

b. The amino acids that can donate carbon atoms to the 1-Carbon pool are Glycine, Serine and Histadine. They all require THF.

6. Indicate the different forms of THF (formyl, methylene and methyl) and reactions requiring the different forms of THF

a. When one carbon is donated to THF you can get formyl THF, methyl THF, and methylene THF. Methyl THF is also the storage form of THF. Degradation of histidine also needs THF. Histidine is degraded to glutamate but in deficiency you will see increased levels of FLGlu in urine after ingesting histidine. Histidinemia.

b. Methylene THF is used as a 1-C donor in the synthesis of purines and thymidinec. Methyl THF is used as a 1-C donor in the conversion of homocysteine to methionine. d. Formyl THF- (Couldn’t find this)

7. Identify the reactions requiring B12 a. Methionine Synthase and methylmalonyl CoA mutase require B12

8. Compare and contrast the causes, clinical and biochemical features of folate and vitamin B12 deficiency

a. Folate acid deficiency (develops in months) is caused by lacking in fruit and vegetables in the diet. It can also be caused by drugs such as methotrexate, trimethoprim (prevent purine and pyrimidine biosynthesis kills blast cells and causes macrocytic anemia).

i. Rapidly growing cancers use folic acid, small bowel malabsorption and sulfa drugs inhibits folic acid synthesis. It is the most common vitamin deficiency in the US and most common in pregnant women and alcoholics. Folic acid before pregnancy reduces neural tube defects. Deficiency results in homocysinuria and megaloblastic anaemia. FIGIu accumulation in blood (histidinemia).

b. Vitamin B12 deficiency (develops in years) causes accumulation of abnormal fatty acids (Cardiovascular disease). Pernicious anaemia because inability to absorb B12

and can lead to megaloblastic anaemia. Causes may be a pure vegan diet, chronic pancreatitis and terminal ileal disease. Increased methylmalonate in circulation.

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9. Indicate the role of intrinsic factor in vitamin B12 absorptiona. Inability to absorb B12 because the parietal cells are injured. So B12 can’t be

absorbed. 10. Justify the mechanism of the occurrence of folate trap in B12 deficiency

a. When you have a B12 deficiency you have a folate deficiency. That is because B12 and methylfolate is required for the homocysteine to methionine reaction however with B12 deficiency all folate gets trapped as methyl THF.

Lecture: Purine metabolism

1. Identify the differences between N-base, nucleoside and nucleotide with examplesa. Addition of a pentose sugar to the nitrogenous base produces a nucleoside. Addition

of either 1, 2, or 3 phosphate groups to a nucleoside produces a nucleoside mono/di/triphosphate. Nucleosides are like Ribose, deoxyribose, cytidine and deoxyadenosine. Nucleotides with be Purines/Pyrimadines.

2. Regarding purine biosynthesisa. Enumerate the C & N donors of the purine ring (amino acids and 1-C groups) (P 13)

b. Outline purine biosynthesis (Draw left P 14 top left)i. Ribose-5-phosphate comes from the HMP pathway. All enzymes necessary

for de novo purine synthesis are found in the cytoplasm of the cell. Before synthesis can take place PRPP is produced from the ribose-5-phosphate and ATP by ribose phosphate pyrophosphokinase. The RLS of purine synthesis is PRPP 5’-phosphoribosylamine and this reaction is catalysed by glutamine PRPP amidotransferase. The rate of this reaction is controlled by high concentrations of glutamine and PRPP. This reaction is also inhibited by the purine nucleotides, AMP, GMP, and IMP. This pathway produces an IMP which can be used to form adenosine (requires GTP) or guanine (requires ATP). The process of creating an IMP molecule requires 4 ATP total and 2 molecules of THF.

ii. The reaction of IMP AMP requires GTP and adenylosuccinate synthetase. The reaction of IMP GMP requires ATP and GMP synthetase. These two Monophosphates are converted to NDP and NTP by nucleoside monophosphate kinases and nucleoside diphosphate kinases respectively.

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c. Discuss the regulation of purine biosynthesis (Identify the regulatory enzyme and positive and negative modulators) (Above)

d. Correlate the mechanism of action of sulfa drugs, trimethoprim, methotrexate, mycophenolic acid to their clinical application

i. Sulfonamides competitively inhibit bacterial synthesis of folic acid. Because purine synthesis requires THF (2 molecules) the sulfa drugs slow down this pathway in bacteria. Sulfonamides are PABA analogues that inhibit the synthesis of folate in bacteria.

ii. Methotrexate is a structural analog of folic acid and inhibits the reduction of dihydrofolate to THF catalysed by dihydrofolate reductase. These drugs limit the amount of THF available for use in purine synthesis and slow down DNA replication in mammalian cells. They are useful in treating rapidly growing cancers (leukemia).

iii. Mycophenolic acid is an inhibitor of inosine monophosphate dehydrogenase. It is a reversible uncompetitive inhibitor of IMP Dehydrogenase. Deprives rapidly proliferating T and B cells of key components of nucleic acids. Drug is used to prevent graft rejection. This doesn’t let you synthesize GMP

e. Explain the formation of deoxyribonucleotides from ribonucleotides (Specify the coenzyme and inhibitor)

i. Reduction is catalysed by ribonucleotide diphosphate reductase. Two important inhibitors are dATP and hydroxyurea (this is why ADA deficiency is bad dATP inhibits ribonucleotide reductase). The coenzyme is Thioredoxin.

3. Regarding salvage pathway of purine bases a. Identify the reactions catalyzed by HGPRT and APRT

i. HGPRT catalyse the reactions of Hypoxanthine IMP and Guanine GMP. ii. APRT catalyzes the reaction of Adenine AMP

b. Correlate the clinical and biochemical features of Lesch Nyhan syndrome (Specify enzyme deficient)

i. It is an X-linked genetic disorder that results in a deficiency of HGPRT. Results in an inability to salvage purine hypoxanthine and guanine. The end product degradation of hypoxanthine and guanine is uric acid. Therefore children with lesch-nyhan have excess uric acid in urine. Orange crystals are found in babies diapers. This disease causes severe mental retardation, self-mutilation (biting lips), and involuntary movement, gout. You find increased PRPP levels and an increase in de novo purine synthesis (this is why you have increased uric acid).

4. Regarding purine nucleotide catabolisma. Explain uric acid formation

i. Purines aren’t essential components of the diet so purines from the diet are degraded to uric acid rather than salvaged.

b. Analyze causes of hyperuricemia, correlate the biochemical basis of the clinical features and explain the biochemical basis of use of allopurinol in the management of hyperuricemia

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i. Deficiency in GALT (metabolize galactose), Glucose 6-phosphotase, and Aldolase B results in hyperuricemia. Gout is characterized by hyperuricemia; acute arthritic joint inflammation caused by deposition of uric acid crystals. Allopurinol is a non-competitive inhibitor of xanthine oxidase. Causes excretion of hypoxanthine and xanthine instead of urate. Better water solubility so excreted from kidney. No buildup of uric acid.

5. Propose the biochemical basis of SCID (ADA deficiency)a. Adenosine deaminase deficiency causes SCIDS. Guanosine and inosine are better

substrates for purine nucleoside phosphorylase than is adenosine. DNA is not synthesized in T-cells and B-cells because of an accumulation of dATP. Extremely large buildups of dATP in red cells.

Lecture: Pyrimidine metabolism

1. Regarding pyrimidine biosynthesisa. Enumerate the donors of C & N atoms to pyrimidine ring (P 22)

b. Outline pyrimidine biosynthesis (UTP & CTP). Distinguish purine and pyrimidine biosynthetic pathways

i. Pyrimidines are synthesized before attachment to the ribose-5-phosphate which is donated by PRPP. The rate limiting step and the committed step for pyrimidine synthesis is the synthesis of carbamoyl phosphate from glutamine and CO2 by carbamoyl phosphate synthetase II (CPS II). CPS II is regulated by it’s products UDP and UTP inhibit it’s activity. Folic acid derivatives are NOT used in pyrimidine synthesis. This pathway creates UMP which can be converted into CTP or dTMP. Utp is converted to CTP by CTP synthase. Glutamine is the nitrogen donor.

c. Summarize the regulation of pyrimidine biosynthesis (regulatory enzymes, regulators) above

d. Explain the synthesis of dTMPi. Thymidylate synthase uses THF to produce dTMP from dUMP. This

reaction is indirectly inhibited by Methotrexate. The reason it is indirect is because this reaction requires THF, methotrexate slows down the synthesis of THF.

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e. Indicate the mechanism of action of 5 fluoro uracil and methotrexate and their clinical application

i. 5-fluorouracil is a uracil analog. The drug is given with thymidine to boost effectiveness. It is converted to dFUMP which inhibites thymidylate synthetase. In cancer cells 5-flurouracil is incorporated into the RNA. This RNA with 5-flurouracil is more detrimental to cancer cells than to normal cells.

ii. Methotrexate is a structural analog of folic acid and inhibits the reduction of dihydrofolate to THF catalysed by dihydrofolate reductase. These drugs limit the amount of THF available for use in purine synthesis and slow down DNA replication in mammalian cells. They are useful in treating rapidly growing cancers (leukemia).

f. Regarding orotic aciduria i. Specify enzyme deficiency and correlate biochemical features to the

clinical features1. A deficiency in Orotate phosphoribosyltransferase (Orotate build

up) and OMP decarboxylase (Orotidine 5’-monophosphate) causes orotic aciduria. Causes abnormal growth, megaloblastic anemia, and excretion of large amounts of orotic acid in the urine.

ii. Compare and contrast orotic aciduria due to defect in pyrimidine biosynthesis and defect in urea cycle

1. An acquired form of the disease may appear in patients being treated for cancer with a pyrimidine analog. A deficiency in these enzymes doesn’t let you create UMP which leads to a build up of their respective precursors (Oratate Orotic Acid). A deficiency in OTC (2nd enzyme) in the urea cycle also leads to orotic aciduria.

2. Indicate the end products of catabolism of the pyrimidine nucleotidesa. Not sure about this one. Unlike purines, pyrimidine rings can be opened and

degraded to highly soluble structures that can serve as precursors for other biomolecules. Pyrimidines can also be salvaged by pyrimidine phosphoribosyltransferase where PRPP is the source of the ribose phosphate.

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Lecture: Serum proteins and associated disorders

1. Describe how plasma proteins can be separated by electrophoresis and classify plasma proteins based on electrophoretic mobility.

a. Serum is preferred for analysis of proteins. Serum proteins can be separated by charge using electrophoresis. The most negatively charged plasma protein and that travels the most is albumin, followed by α-1, α-2, beta and then gamma proteins.

2. Describe the functions of serum albumin and globulins. a. Albumin- most abundant serum protein and contributes to the osmotic pressure of

the plasma. It cannot cross the capillary wall into the interstitial space and it helps to retain water in the intravascular space. When serum albumin levels are low there is an increased amount of water in the interstitial space leading to edema. Another effect is low colloid osmotic pressure. Individuals with congenital analbuminemia appear normal. Albumin can be glycosylated at high serum glucose levels (could use for a diabetes text). Presence of albumin in urine implies damage to basement membrane of the glomerulus. Albumin since it is so negative can bind Calcium. An increase in plasma pH results in higher binding of calcium to albumin resulting in hypocalcemia. Albumin also transports free fatty acids in it’s hydrophobic pockets. It can also bind hormones like thyroxine and serve as a reservoir for free hormone. Albumin binds bilirubin as well and transports it to the liver for conjugation, however drugs (aspirin) can displace bilirubin from albumin and lead to kemicterus in infants (bilirubin accumulation in the brain).

b. Globulins- Transport, enzymes and inhibition of proteases (more specific below Q. 5)3. Indicate the role of proteins of the complement system (Couldn’t find in notes)4. Predict three common causes of hypoalbuminemia (liver disease, nephrotic syndrome,

protein malnutrition) and explain the biochemical basis for the occurrence of edema in hypoalbuminemia

a. Decrease synthesis of albumin is a result of a low protein diet (malnutrition) or chronic liver disease (cirrhosis). Increased loss of albumin can be a result of nephrotic syndrome where albumin is lost via the urine.

5. Distinguish the functions of proteins that are found in the a. α1-globulin fraction (α1- antitrypsin, α fetoprotein, retinol binding protein,

transcortin)1. α1-antitrypsin- an inhibitory protein against neutrophil elastase in alveoli. A

deficiency of α1-antitrypsin permits neutrophil elastase to destroy lung. May lead to emphysema. This protein is normally synthesized in the liver and is N-glycosylated. M allele is the normal allele and Z/S allele are defective alleles. Individuals who are homozygous for the Z allele (Pi ZZ) have high risk of developing pulmonary and liver disease. Smoking will increase the risk for emphysema because smoke oxidizes methionine in α1-antitrypsin which is needed to bind elastase.

2. Α fetoprotein (AFP)- is abundant in fetal plasma and may have similar function to albumin. High maternal serum AFP indicates fetal neural tube

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defects. Low maternal serum AFP is an indicator of Down syndrome. AFP in adults is used as a marker for hepatocellular cancers.

3. Transcortin- synthesized in the liver and in blood and is the main transport protein for cortisol

4. Retinol binding protein (RBP)- transports retinol in blood. Delivers retinol from the liver stores to the peripheral tissues.

b. α2-globulin fraction (α2 macroglobulin, haptoglobin, ceruloplasmin)1. α2 macroglobulin is one of the largest serum proteins and acts as protease

inhibitor. It binds to and inactivates proteases like plasmin and thrombin. Levels of α2 macroglobulin are elevated in nephrotic syndrome. Loss to urine is prevented by it’s large size

2. Haptoglobin- A serum protein that binds to free hemoglobin in circulation. Haptoglobin-Hemoglobin complex cannot be excreted by the kidney, thus preventing loss of hemoglobin (iron and globin). Low serum free haptoglobin levels are found in patients with acute hemolysis (haptoglobin-hemoglobin complexes increase) and can be used to monitor patients with hemolytic anemia.

3. Ceruloplasmin- A copper containing plasma protein and is synthesized and secreted by the liver. Ceruloplasmin has ferroxidase activity and helps in the oxidation of Fe2+ to Fe3+ so it can be encorporated into transferrin. Low ceruloplasmin levels are found in wilson’s disease. Due to less attachment of copper to ceruloplasmin, copper accumulates in tissues “Kayser-Fleischer rings.”

c. β-globulin fraction (transferrin, hemopexin, β lipoproteins)1. Transferrin is a transport protein for iron which needs to be in the ferric

form. Trasnsferrin transports iron between intestine, liver, bone marrow and spleen. Each transferrin can bind 2 Fe3+. Patients with iron deficiency have low transferrin saturation.

2. Hemopexin- Binds to free heme in circulation and prevents the loss of iron by the kidneys.

3. LDL/β-Lipoproteins- Most negatively charged because of apo-b-100. Transport cholesterol.

d. γ-globulin fraction (Immunoglobulins: Ig G, M, A, D, E)1. Are immunoglobulins produced by activated B lymphocytes (plasma cells)2. IgM- first antibody to be produced in response to an antigen3. IgG- antibody produced on repeated exposure to same antigen. It can also

cross the placenta and confer immunity to the fetus and newborn4. IgE- secreted in response to an allergen

6. Explain the consequences of inherited α1-antitrypsin deficiency on the liver and the lungs and discuss the effects of smoking on the structure of 1-antitrypsin.

a. Individuals who are homozygous for the Z allele (Pi ZZ) have high risk of developing pulmonary and liver disease (accumulates in liver). Smoking will increase the risk for emphysema because smoke oxidizes methionine in α1-antitrypsin which is needed to bind elastase.

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7. Interpret the utility of C-reactive protein as an inflammatory markera. Acute phase proteins are increased in any inflammatory disorder. Cytokines released

during inflammation stimulate hepatic synthesis of these proteins. Typical examples include C-reactive protein, ceruloplasmin, and haptoglobin. CRP levels are measured by specific tests to monitor the progress of an inflammatory reaction.

8. Recognize the serum protein electrophoretic pattern in the following diseases (hypoalbuminemia, α1-antitrypsin deficiency, polyclonal gammopathy, monoclonal gammopathy) Draw out all the charts and say what is elevated on page before blank

Lecture: Heme synthesis and porphyrias (Draw last page out with Lippincott additions)

1. Describe in detail ALA synthase and ALA dehydratase reactions.a. ALA Synthase- It has two isozymes. ALAS1 for the liver and ALAS2 for the RBC. They

are regulated in different ways. ALA synthase uses a succinyl-CoA and glycine to create ALA. ALA synthase needs PLP as a coenzyme as it decarboxylates Glycine.

i. ALAS1- Drugs increase ALAS1 activity as they lead to CYP 450 synthesis which needs heme. Low intracellular heme concentration leads to ALAS1 synthesis. Heme synthesis stops when heme is not incorporated into proteins and as result, heme and hemin accumulate. Hemin decreases the synthesis of ALAS1 and also the mitochondrial import of the enzyme from cytosol. Down-regulated by heme/hemin acumulation

ii. ALA Dehydratase (Porphobilinogen Synthase)- Accumulation of heme will lead to MORE globin chain synthesis. Synthesis of heme in eythroid cells is under control of erythropoietin and availability of intracellular iron. ALAS2 is found in fetal liver and adult bone marrow and a deficiency leads to X-linked sideroblastic anemia. Down-regulated at low amounts of intracellular iron.

2. Outline heme synthesis starting from glycine and succinyl CoA to the formation of hemea. Refer to drawing on left

3. Distinguish between the different regulatory processes of heme synthesis in the liver and in the erythroid cells. (Question 1)

4. Explain how pyridoxine deficiency affects heme synthesis.a. PLP is needed as a coenzyme for ALA synthase and heme synthesis cannot start if

this is deficient. 5. Discuss the effects of lead poisoning on heme synthesis.

a. This metal can interact with the zinc cofactors for ALA dehydratase and ferrochelatase. ALA and protoporphyrin IX accumulate in urine. Ferrochelatase needs zinc as well as ALA hydratase (porphobilinogen synthase). Leads to accumulation of ALA and protoporphyrin IX.

6. Categorise the different types of porphyrias based on clinical manifestations such as abdominal pain, photosensitivity and neuropsychiatric symptoms.

a. Acute Intermittent Porphyria- Deficiency of HMB Synthase. ALA and porphobilinogen in blood and urine. ALA activity is high because ALA synthase is not feed-back inhibited by heme or hemin (none is being created) so the synthesis of the ALA synthase enzyme is increased. AIP can be diagnosed by urine color because on

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exposure to light and air it changes from the colorless porphobilinogen to dark colored urine (porphobilin). Symptoms include very severe abdominal pain, high agitate state, mental disturbance, weakness of lower extremeties. Patients are NOT photosenstivive as HMB cannot be formed. ALA and porphobilinogen can act as neurotransmitters. You do not administer barbiturates to these patients because it stimulates the synthesis of CYP450 and that can worsen the situation.

b. Congential Erythropoietic Porphyria (Does not effect liver)- Deficiency of Uroprophyrinogen III Synthase. Autosomal recessive trait that is characterized by extreme photosensitivity and reddish brown teeth with werewolf features. Hemin infusion would not help because erythroid cells contain ALAS2 which is not inhibited by hemin rather it is stimulated by high intracellular concentration of iron. You see Uroporphyrin I and Coproporphyrin I in the urine.

c. Porphyria Cutanea Tarda- caused by a deficiency in uropophyrinogen decarboxylase. Uroporphyrin accumulates in the urine and it is the most common porphyria and patients are photosensitive. Type I is sporadic and Type II is familial. Accumulation of uroporphyrin in the skin and also the liver. Erosions and bullous lesions in sun-exposed areas of the patients are produced by minor trauma because of increased skin fragility. Urine is red upon release due to high levels of red uroporphyrin III.

7. Describe the biochemical basis for the clinical features (abdominal pain and photosensitivity, dark colored urine) occurring in acute intermittent porphyria, congenital erythropoitic porphyria and porphyria cutanea tarda. Indicate the deficient enzyme and products accumulating in the above mentioned disorders. (read above it’s all there)

Lecture: Heme degradation and jaundice

1. Outline the steps in the degradation of heme to bilirubin in the macrophages.a. RBC half a lifespan of about 120 days. Old RBCs are sequestered by the spleen. RBCs

contain hemoglobin that is broken down to heme and globin. Heme Oxygenase converts heme to biliverdin (green pigment). This reaction also leads to the porphyrin ring being cleaved, only reaction in body that creates Carbon monoxide and converted the ferrous iron to ferric (3+).

b. Next the biliverdin is acted upon by bilverdin reductase which forms bilirubin (orange/yellow color). This is known as unconjugated bilirubin or free bilirubin.

2. Explore the relationship of serum albumin and the transport of bilirubin a. Bilirubin formed in macrophages is not water soluble. It binds in blood to albumin

for transport. This bilirubin is known as unconjugated bilirubin. Binding of bilirubin to albumin prevents it from being excreted in urine. Many drugs can displace bilirubin from albumin and this can lead to a lot of free bilirubin that can cross blood brain barrier causing kenicterus in children. This unconjugated bilirubin is the taken up by the liver by specific transporters and is bound to the intracellular liver protein ligandin.

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3. Explain the biochemical consequence of a block in each of the steps in the uptake and conjugation of bilirubin in liver. Distinguish the activity of UDP-glucuronyl transferase in adults versus premature babies.

a. Bilirubin is converted to conjugated bilirubin by the addition of two molecules of glucuronic acid. The enzyme is microsomal UDP glucuronyl transferase creating conjugated bilirubin. If you block the uptake of bilirubin into the liver you will have high circulating levels of unconjugated bilirubin. If you block conjugation of bilirubin you will have kernicterus in children (specific defects discussed later).

b. Newborn infants have low activity of hepatic UDPglucuronyl transferase (premature have even lower activity). You have jaundice by 2nd or 3rd day of life that usually clears by the 7th day of life. Increased destruction of RBCs after birth overloads the liver’s capacity to conjugate bilirubin.

4. Analyze the steps in the processing of conjugated bilirubin in the intestine and its excretion in feces and urine

a. After bilirubin has been conjugated it is actively transported into the bile canaliculus against a concentration gradient by a specific ABC transporter. Conjugated bilirubin is a component of bile and is released into the second part of the duodenum via the common bile duct. Conjugated bilirubin is acted upon by bacterial flora in the large intestine. Conjugated bilirubin undergoes deconjugation and is converted to urobilinogen (colorless). Bacterial action on urobilinogen forms stercobilin (brown) that is lost in the feces and gives the feces it’s brown color. Some urobilinogen is absorbed from the gut into portal blood. A majority of urobilinogen is lost in the urine as urobilin (light yellow color).

5. Identify the sequential steps in heme catabolism beginning with heme till the formation of urobilinogen and stercobilin; identify the various tissues/organs involved in each step

a. (Refer to drawing on left as well). Heme is converted to biliverdin by Heme oxygenase in the macrophage. The biliverdin is then converted to bilirubin by bilverdin reductase once again in the macrophage. (This process happens in reticuloendothelial system)

b. The free bilirubin is the transported in the blood by albumin which transports it to the liver for conjugation. At the liver the bilirubin is taken up by specific receptors and is bound intracellularly to a protein called ligandin.

c. Within the liver UDP glucoronyl transferase adds 2 mols of glucuronic acid via UDP-glucuronic acid. The conjugated bilirubin is the actively transported into the bile canaliculus against a concentration gradient by a specific ABC transporter.

d. Conjugated bilirubin is a component of bile and is released into the second part of the duodenum via the bile duct. Conjugated bilirubin is acted upon by bacterial flora converted into the colorless urobilinogen. Urobilinogen can be converted to stercobilin giving feces the brown color. It can also be reabsorbed by the portal system and sent back to the liver. However a majority is lost in urine as urobilin (light yellow).

6. Compare and contrast conjugated and unconjugated bilirubin with reference to chemical composition, water solubility, tissue deposition, excretion in urine and common clinical conditions where they are elevated

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a. Conjugated bilirubin is water soluble. You have high levels of unconjugated bilirubin in kernicterus, prehepatic jaundice, and hepatic jaundice. You have elevated levels of conjugated bilirubin in hepatic jaundice and post hepatic jaundice. Free bilirubin is not excreted in urine because it is bound to albumin. Conjugated bilirubin can be converted to urobilinogen which can be converted to urobilin (urine) or stercobilin (feces).

7. Distinguish conjugated and unconjugated hyperbilirubinemia theoretically using lab testsa. Van den Bergh reaction- Bilirubin reacts with diazo reagent to form a red colored

complex. Conjugated bilirubin reacts rapidly with the reagent and is called direct bilirubin.

b. Total bilirubin- the reaction is also carried out in the presence of methanol because unconjugated bilirubin is water insoluble and reacts in the presence of methanol. (Indirect reacting)

c. Total bilirubin – Direct (conjugated) bilirubin = indirect (unconjugated) Bilirubin. d. Hyperbilirubinemia is characterized by serum bilirubin > 2 mg/dL

8. Explain the biochemical mechanisms involved in the development of physiological jaundice of the newborn

a. Newborns have a low activity of hepatic UDP glucuronyl transferase. This leads to less conjugated bilirubin and more free bilirubin in the serum. This is caused by an increased destruction of RBCs after birth which overloads the livers capacity to conjugate bilirubin (high unconjugated bilirubin levels in newborn). Unconjugated bilirubin can now corss the blood brain barrier and deposit in the basal ganglia of the brain resulting in kernicterus as it is lipid soluble. (kernicterus can also be caused by drugs such as salicylates, sulfonamides that compete with bilirubin for albumin binding increasing the amount of unconjugated bilirubin in the blood). This disorder is characterized by severe neurological symptoms.

9. Explain the rationale of phototherapy and phenobarbital in the therapy for premature babies with jaundice.

a. Phototherapy in neonatal jaundice in premature babies with jaundice is helpful because light converts bilirubin to more polar, water soluble isomers, that can be excreted in bile without conjugation.

10. Distinguish prehepatic, hepatic and post hepatic jaundice based on lab data.

ConditionSerum total

Bilirubin

Serum conjugated

Bilirubin

Serum unconjugated

Bilirubin

Urine Bilirubin

Urine Urobilinogen

Prehepatic Jaundice

N Absent

Hepatic Jaundice

Present N or or

Posthepatic Jaundice

(Obstructive) N Present /Absent

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11. Interpret laboratory data in the different types of jaundice (Look at table with drawings)a. Prehepatic: sickle cell anemia, G6PD deficiency (Write disorders by respective

drawings)b. Hepatic: alcoholic cirrhosis, hepatitisc. Obstructive jaundice due to gall stones or cancer of head of pancreas

12. Differentiate between inherited disorders: Gilbert’s syndrome and Crigler-Najjar syndromes I and II based on the pathogenetic mechanisms and biochemical alterations

a. Crigler Najjar Type I- most severe and is associated with an almost complete deficiency of the enzyme bilirubin glucuronyl transferase. Serum bilirubin levels acan reach upto 50 mg/dL and result in jernicterus and mental retardation. Management of crigler-najjar syndrome type I is daily phototherapy.

b. Crigler Najjar Type II- (Arias syndrome) has a lower activity of bilirubin glucuronyl transferase (10-20% of normal). Characterized by jaundice but less severe than type I. Children respond to phenobarbital (induces the enzyme). Regular phototerapy is also used.

c. Gilbert’s syndrome- present in 3-7% of the population and is the most common. Characterized by mild jaundice following stress or starvation. UDP glucuronyl transferase activity is about 50% of normal. Mild increase in unconjugated bilirubin.

d. Dubin-Johnson syndrome- inherited deficiency of the ABC transporter that transports conjugated bilirubin from the hepatocyte into the biliary canaliculus. Characterized by elevated levels of conjugated (direct) bilirubin in circulation.

Lecture: Hemostasis

1. Outline the four phases of hemostasis.a. Vascular Spasm/Vasoconstrictionb. Platelet plug formation/primary hemostasisc. Blood coagulation/secondary hemostasisd. Dissolution of the fibrin clot/tertiary hemostasis

2. Discuss vascular spasm and the role of endothelin.a. Trauma to the vessel wall results in smooth muscle contraction. Contraction is

caused by local myogenic spasm, factors released from the injured vessel wall (endothelin and serotonin) and nervous reflexes. This cannot cause long term cessation of bleeding.

3. Describe the platelet plug formation.a. Greatly limits the loss of blood from the circulation by forming plugs. Small cuts in

the blood vessels are often sealed by platelet plugs. Platelets first adhere to the damaged surface and activate. Then you have platelet aggregation.

b. Platelet adhesion and activation is facilitated by endothelial injury. Platelet adhesion is mediated by platelet receptors glycoprotein Ib/Ia. These receptors bind to ligands that are components of the subendothelial matrix (collagen, von Willebrand Factor)

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c. Platelet GpIa binds to collagen. Leads to development of pseydopods to promote platelet-platelet interactions. vWF binds to the platelet receptor glycoprotein Ib resulting in changes in the platelet membrane. This binding also exposes GPIIb/IIa for binding of fibrinogen.

d. vWF acts as a bridge between specific glycoproteins (GP Ib) on the surface of platelets and collagen fibers. It facilitates platelet adhesion to the vessel wall and platelet aggregation. Complexes with factor VIII; carries it, stabilizes it and prevents it’s degradation. vWF deficiency is associated with a defect in the formation of the platelet plug (primary hemostasis)

4. Explain the role of integrins GPIb and GPIa. (Question above)5. Describe the role of platelet-activating factors (ADP, serotonin, TXA2 and thrombin).

a. ADP- released from the platelet granules. It is a potent platelet activator, it promotes platelet-platelet contact, and platelet adherence. Binding of ADP to it’s receptor facilitates the release of Ca2+ and decerases cAMP levels (both of which facilitate platelet aggregation). Binds to receptor on platelet membrane leading to further unmasking of GpIIb/GpIIIa binding sites which bind fibrinogen.

b. Once ADP binding releases Ca2+ phospholipase A2 is stimulated and it creates TXA2

from arachidonic acid. This leads to platelet aggregation by activating other platelets. Diffuses out of the cell to enhance vasoconstriction.

c. Serotonin- function sin the same way as endothelin. Contracts the vessel in response to trauma. Part of the vascular spasm phase of hemostasis.

d. Conversion of fibrinogen to fibrin requires thrombin. Fibrinogen is a plasma protein that is synthesized by the liver and cleaves the fibrinopeptides of fibrinogen to form the fibrin monomer. The fibrin monomers aggregate and are linked to each other via hydrogen bonds, forming the fibrin polymer (soft clot). Formation of a hard clot is mediated by factor XIII activated by thrombin.

6. Describe the role of integrin GPIIb/IIIa in platelet aggregation. (Above)7. Describe the coagulation cascade, including the extrinsic, intrinsic and common pathways.

a. Aim is to convert soluble fibrinogen to insoluble fibrin (threads that stabilize the platelet plug) Requires thrombin.

b. Extrinsic (draw these out)i. Tissue Injury leads to release of tissue factor (factor III or

thromboplastin). ii. Factor VII is activated by tissue factor to VIIa.

iii. Factor VIIa and tissue factor III in the presence of Ca2+ and platelet phospholipids activate factor X

iv. Xa (active factor X)c. Intrinsic

i. Rough endothelial surface exposure of collagen activation of factor XIIii. XIIa acts on XI to activate it

iii. Xia acts on IX to activate itiv. Thrombin activates factor VII VIIav. IXa, VIIIa, platelet phospholipids and Ca2+ activate factor X to Xa

d. Common Pathway

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i. Thrombin acts on factor V Vaii. Xa combines with Va, tissue phospholipids and Ca2+ to form prothrombinase

complex. iii. Prothrombinase complex splits prothrombin to form thrombiniv. Thrombin acts on fibrinogen to fibrinv. Thrombin activates factor XIII to XIIIa

vi. Fibrin monomers are covalently crosslinked by factor XIIIa to form cross-linked fibrin

e. Thrombin production from prothrombin needs factor Xa. Ultimate aim of hemostasis is conversion of fibrinogen to fibrin and stabilization of fibrin which requires thrombin IIa

8. Indicate the role of calcium in hemostasis (Above)9. Discuss the role of vitamin K in the post-translational modification of clotting factors

a. Vitamin K is required for the hepatic synthesis of prothrombin (factor II), VII, IX, X, protein C and S. Vitamin K dependent γ-decarboxylation of glutamic acid residues of the above proteins. This process forms mature clotting factors and is capable of activation. This carboxylation allows Ca2+ binding because of two adjacent negatively charged carboxylate groups. Clotting factor Ca2+ complex can then bind to phospholipids on the platelet membrane.

10. Predict the clinical manifestations in a patient with vitamin K deficiency a. The extrinsic and intrinsic pathways will not work because the factors won’t be

synthesized. 11. Discuss the regulation of coagulation, and fibrinolysis; outline the role of PGI2,

antithrombin III, protein C, TFPI, t-PA and plasmin. a. PGI2- prevents platelet aggregation. Increases cAMP levels within platelets and

inhibits platelet activation. It is a thromboxane antagonist. Coagulation automatically initiates fibrinolysis. Hemostasis controller.

b. Antithrombin III- binds to and inhibits factor Xa and thrombin (IIa). Heparin acts by activating this factor and preventing coagulation

c. Protein C and S- require vitamin K for γ-decarboxylation. Act together to inactivate cofactors Va and VIIIa. Protein C is activated by binding of thrombomodulin to thrombin. Protein S is a cofactor for protein C. Prevents coagulation.

d. Plasmin- Inactive plasminogen gets incorporated in the developing clot. Once it is activated it turns into plasmin which is proteolytic. It degrades fibrin to fibrin degradation products. (Dissolutes the fibrin clot)

12. Identify the role of bleeding time, clotting time, prothrombin time (INR), and activated patial thromboplastin time (APTT) in the differentiation of bleeding disorders.

a. Hemostatic function testsi. Bleeding time- test for the time taken from the initial injury to the formation of

the primary hemostatic plug. Bleeding time is an indicator of primary hemostasis. Prolonged bleeding time is an indicator of low platelet count or vWF deficiency or platelet receptor defects.

ii. Clotting time- time taken for the formation of the stable hemostatic plug. Prolonged clotting time indicates defects in the coagulation pathway. Specific

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defects of the instrinsic/extrinsic pathways are indicated by prothrombin time and activated partial thromboplastin time (APTT/PTT)

b. Tests for Coagulation Disordersi. Prothrombin Time (PT)- tests the extrinsic and the common coagulation

pathways. International normalized ratio (INR). Measures defects in tissue factor, VII, V, X, prothrombin and fibrinogen (extrinsic)

ii. Partial thromboplastin time- (APTT)- Tests intrinsic and common pathways. Measures defects in VIII, IX, XI, XII, V, X prothrombin and fibrinogen.

13. Discuss the biochemical basis of the following genetic disorders of hemostasis: hemophilia (A and B), Von Willebrand Disease, Bernard-Soulier Syndrome, Thrombasthenia of Glanzmann and Naegeli.

a. Hemophilia- inherited X-linked recessive disorder. They have a coagulation factor deficiency. Easy bruising, massive hemorrhage after trauma and surgical procedures. Spontaneous hemorrhages, particularly in the joints- hemoarthrosis. Clotting time is increased and APTT is increased (intrinsic pathway). Type A (VIII) and Type B (IX).

b. Von Willebrand Disease- Most common inherited bleeding disorder. Instability of factor VIII. Similar features to hemophilia A, with increased mucosal bleeding, increased post operative bleeding. Bleeding time is prolonged, APTT prolonged and vWF levels are low.

c. Bernard-Souller Syndrome- Qualitative defect of platelets. Normal platelet count but increased bleeding time. GpIB defect.

d. Thrombasthenia of Glanzman and Naegeli- Qualitative defect of platelets. Normal platelet count but increased bleeding time. GpIIb/IIIa defect.

e. Thrombocytopenia- Low platelet count and increased bleeding time.

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Lecture: Liver function tests

1. List the important functions of the liver a. Excretion of bilirubinb. Synthesis of plasma proteinsc. Detoxification of ammonia

2. Interpret the values of the following laboratory tests in the diagnosis, follow up and prognosis of a patient with liver disease (acute hepatitis, alcoholic liver disease, cholestatic disease)

a. Serum (total, conjugated and unconjugated) and urine bilirubin1. In Patients with hepatitis, serum total, conjugated and unconjugated bilirubin

is increased. Damaged liver has a lower than normal capacity for uptake and conjugation of bilirubin. Conjugated bilirubin may also be elevated due to inflammation because the liver cell swells and blocks the ductules.

2. In Cholestatic disease serum conjugated bilirubin is elevated. In complete obstruction conjugated bilirubin is not excreted in the bile and it regurgitates into blood and appears in the urine. Urobilnogen is also not formed resulting in clay colored feces (stercobilinogen cannot be formed either). Serum bilirubin can estimate the extent of the damage but it may be difficult to distinguish hepatocellular disorder from a cholestatic disorder on basis of bilirubin itself.

b. Serum enzymes (ALT, AST, GGT, 5’NT, LDH) Allow you to distinguish hepatocellular or cholestasis

1. Hepatocellular- ALT and AST are enzymes related to amino acid metabolism in the liver. Levels are raised in viral hepatitis, drug induced hepatitis, and long standing obstructive jaundice. Raised ALT is more specific for liver cell damage compared to raised AST. In acute hepatitis ALT >>> AST. In long standing (chronic) alcohol cirrhosis AST >>> ALT.

2. Cholestasis- ALP and GGT. GGT synthesis is induced by alcohol and this enzyme is a marker of alcohol consumption. ALP and GGT are both elevated in biliary stasis (Cholestasis). If biliary tree is dilated there is extrahepatic cholestasis, if it is not dilated then intra-hepatic cholestasis is suspected. ALP increase with no GGT increase indicates that it is a non-hepatic cause. Elevation in GGT and not ALP indicates recent alcohol use.

c. Serum proteins (albumin and globulin)1. All plasma proteins except the γ-globulins are synthesized by the liver. Albumin

helps in maintenance of colloidal osmotic pressure. Chronic lever disease is characterized by low serum albumin levels. Hypoalbuminemia leads to ascites and edema in patients. Decrease of albumin indicates long standing liver disease. Serum γ-globulins are increased in cirrhosis.

d. Prothrombin time1. Liver synthesizes factors V, VII, IX, X, prothrombin and fibrinogen. Normal

prothrombin time is 12-15 seconds. Increased time indicates deficiency of clotting factors synthesized by the livers. May be due to hepatocellular disorder due to decreased synthesis of clotting factors. May also be elevated

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due to cholestasis. Cholestasis results in an impaired absorption of vitamin K due to reduced entry of bile acids into intestine. This impairs post translational γ-carboxylation modification of vitamin K clotting factors II, VII, IX and X.

e. Special tests in evaluating liver function: Serum ammonia, AFP, α1- antitrypsin, ceruloplasmin, serum iron, transferrin and ferritin

1. Serum ammonia levels contribute to the development of hepatic encephalopathy and altered consciousness in patients with end stage liver disease. Impairment of urea formation by the liver and [blood ammonia] rises.

2. Serum alpha fetoprotein (AFP) is used as a tumor marker in patients with liver cancer.

3. Serum iron, transferrin and ferritin is used in patients suspected to have hemochromatosis

4. Serum ceruloplasmin- patients suspected to have Wilson’s disease (copper)5. Serum α1-antitrypsin in patients suspected to have hereditary disease of the

enzyme. Leads to emphysema. ___________Read the Cases at the end of the lecture for these last three___________3. Compare and contrast the changes in liver function tests in hepatocellular diseases and

disorders associated with cholestasis4. Differentiate between acute and chronic liver disease based on liver function tests5. Explain the biochemical mechanisms for the following symptoms and signs in patients

with liver disease – edema, icterus, ascites, encephalopathy, bleeding tendency

Lecture: Alcohol and xenobiotic metabolism in liver

1. Explain the basic mechanisms of drug metabolism.a. Drug metabolism in the liver can add or expose a functional group that makes

molecule more polar for eventual excretion in the kidney. 2. Define phase I and phase II reactions.

a. Phase I involves cytochrome P450 (CYP450) and is in ER membranes. Cytochrome P450 enzymes are involved with phase I which leads to hydroxylations or other reactions. Cytochrome P450 enzymes use molecular oxygen and need NADPH and a heme group. Cytochrome P450 reductase and cytochrome P450 work together.

b. Phase II follows in many cases phase I but some drugs are also changed into more water soluble forms and occurs in the cytosol. Phase II uses mainly UDP-glucuronic acid, PAPS for sulfation, glutathione and amino acids.

3. Describe the role of cytochrome P450 enzymes in drug metabolism and specify CYP2E1 and CYP 3A4. CYP stands for Cytochrome P450

a. Some drugs are inactivated by CYP450. Some prodrugs need activation by CYP450 in order to be in their active form. Some drugs may be converted to a toxic metabolite following the action of CYP450.

b. CYP3A4 is one third of CYP450 in the liver and acts on more than half of the therapeutic drugs.

c. CYP2E1 is specific for ethanol metabolism and is part of the MEOS system in the liver.

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4. Describe the mechanism of acetaminophen toxicity and the biochemical basis for its management. (Page 42)

a. Acetominophen is designed to be mostly metabolized by the liver to acetaminophen glucuronate and to acetaminophen sulfate in order to be excreted by the kidney. If CYP2E1 is induced by chronic alchohol abuse then this pathway can be increased and can lead to a high amount of NAPQ I. Even then NAPQ I can be excreted by the kidney after linkage to glutathione. At toxic high levels NAPQ I can lead to cell death and sever liver damage especially when GSH is not available in sufficient quantity for detoxification. Patients with acetaminophen poisoning can be trated by administration of N-acetyl-cysteine (acetadote). This drug binds to NAPQ I and makes it more water soluble and the cysteine part of the drug allows synthesis of more GSH.

5. Describe the routes for ethanol metabolism. Discuss the role of alcohol dehydrogenase, acetaldehyde dehydrogenases, and the microsomal ethanol oxidizing system in the metabolism of ethanol. (P 43)

a. Uptake of ethanol starts in the stomach. The liver is the major organ to metabolize most of the blood ethanol to acetaldehyde and then to acetate. Acetaldehyde is toxic and can be formed by alcohol dehydrogenase and at very high ethanol concentrations by MEOS and small amount by catalase.

b. At low alcohol levels in the liver Alcohol dehydrogenase converts ethanol to acetaldehyde and creates an NADH. Alcohol dehydrogenase have a low Km for ethanol.

c. At high ethanol levels the MEOS system which also generates cytosolic acetaldehyde is also active. MEOS has a large KM and this also creates radicals that can’t be scavenged.

d. Acetylaldehyde that is formed in cytosol enters mitochondria. Mitochondrial ALDH-2 has a smaller Km than cytosolic ALDH-1. These reactions convert the toxic acetaldehyde to acetate and form an NADH.

6. Outline the fate of acetate formed from ethanola. The acetate is released into the blood by the liver. The acetate is used mainly in

muscle and heart to form acetyl-CoA catalyzed by acetyl-CoA synthetase. This is an irreversible reaction and the formed acetyl-CoA enters the TCA cycle.

7. Discuss the physiological relevance of induction of CYP2E1 by ethanol.a. CYP2E1 has a lower affinity of ethanol than liver alcohol dehydrogenase. This means

you need a large amount of alcohol to induce this enzyme. This enzyme generates more radicals than normal in the cytosol. In addition the high acetaldehyde levels caused by the large intake of alcohol leads to acetaldehyde binding to glutathione and impairs scavenging of ROS. The increase in radical formation by MEOS (CYP2E1) leads to damage of mitochondrial DNA and proteins and general cell damage.

8. Describe the biochemical basis for the acute and chronic toxic effects of ethanol abuse.a. In chronic alcoholic individual, the high NADH levels lead to increased synthesis of

TAGs and VLDL formation. This happens after a meal and also during fasting. Lactate cannot be used to form pyruvate and the lactate stays in the blood. Pyruvate is lost

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as lactate. Glucogenic amino acids cannot be used as OAA cannot be formed from malate in cytosol. Glycerol-3-P cannot be used to form DHAP in cytosol.

9. Discuss the treatment of poisoning by methanol or ethylene glycol using ethanol.a. Both poisons are substrates in cytosol for alcohol dehydrogenase. Treatment can

include in both cases inhibition of alcohol dehydrogenase by ethanol or a competitive drug (fomepizole).

b. Methanol poisoning can be treated using ethanol or medical drugs to compete with methanol as substrate for alcohol dehydrogenase. Alcohol dehydrogenase has a higher affinity for ethanol than for methanol. Methanol can lead to formaldehyde which can result in blindness in severe cases.

c. Ethylene glycol is an alcohol found in antifreeze and when ingested alcohol dehydrogenase forms glycoaldehyde. Can lead to kidney failure and death due to formation of calcium oxalate. Treatment includes emptying of stomach and patients need to be on ICU>

10. Describe the action of drugs that inhibit aldehyde dehydrogenase (Disulfiram) (Page 47)a. Acetaldehyde dehydrogenase, especially the mitochondrial form, is inhibited by

disulfiram (antabuse). The levels of acetaldehyde increase as cytosolic acetaldehyde dehydrogenase has a larger Km. Acetaldehyde can accumulate to levels that lead to flushing and vomiting. Disulfiram treatment should prevent intake of even low amounts of alcohol in these patients, but it can be also dangerous when the patient drinks large amounts of alcohol (in spite of disulfiram) and the acetaldehyde levels can increase dramatically to toxic levels.

Lecture: Intertissue relationships: Adipose Tissue, Muscle and Brain Metabolism (2 lectures)

1. Develop the concept that the metabolism of the body is influenced by the liver, adipose, muscle and brain, as well as other tissues (This is dumb I’m not answering it)

2. Recognize the 5 major functions of adipose tissue and differentiate between the two general types of adipose tissue (white and brown)

a. Energy storage (Discuss TAG as a concentrated form of metabolic energy)b. Protection of internal organsc. Insulation of body heatd. Adipose as an endocrine gland

i. Discuss leptin as an adipokine- Leptin receptors are in the hypothalamus. Product of the Ob gene (obesity) and tells brain you’ve eaten enough. Leptin induces expression of UCP-2. Uncoupling proteins are found in the inner mitochondrial membrane. UCP form channels which allow dissipation of the proton gradient. Contributes to heat production. Leptin also activates AMPK (AMP-dependent kinase).

ii. Discuss leptin’s role in appetite regulation- Mediates appetite suppression. iii. Link leptin resistance to insulin resistance- In obese patients leptin levels are

high and there is no signal pathway to stop the person from eating. The signaling pathway is messed up. Likewise in insulin resistance there is an

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absence of signal response so it results in high insulin after a meal. The high insulin is constant because the person keeps eating and the leptin is being produced to tell the person to stop, however the receptors are defective so no signal is being sent.

e. Thermogeneration (Brown Adipose Tissue, BAT)i. Discuss the cellular characteristics of BAT- more mitochondria and many

dispersed fat droplets. ii. Function of the H+ uncoupler thermogenin (UCP1)- Allows thermogenesis and

works by dissipating the proton gradient in the mitochondria. Forms pores in the membranes.

3. Review TAG hydrolysis and the synthesis of TAG in adipose tissuea. Relate to the fed/fast state- Fasting state hormone sensitive lipase will be activated

by epinephrine and glucagon. There is a decrease in insulin. In the fed state HSL will be inhibited by insulin.

b. Review sources of FA for the adipose cell: Chylomicrons and VLDL- Occurs in a well fed state. High insulin/glucagon ration leads to an increase in GLUT-4 transporters. Glucose uptake into the liver. VLDL is synthesized in the liver and released into the blood. Lipoprotein lipase of adipocyte is activated by insulin. Cleaves TAGs in VLDL and CM (Apo-C-II).

4. Review the possible sources of glycerol-3-phosphate in the adipose and the liver and discuss the fate of the glycerol that is released from adipose tissue TAG lipolysis

a. Glycerol that is released from the adipose tissue by TAG lipolysis goes to the liver where is converted to glycerol 3-P by glycerol kinase which can be converted to DHAP Gluconeogenesis. In the adipose tissue Glucose is taken in by the cell and converted to DHAP which is converted to Glycerol 3-P and is used in TAG synthesis.

5. Associate abdominal fat with increased risk of coronary heart disease (Lippincott’s 5 th

Edtion – p350)a. Visceral fat is located inside the abdominal cavity packed in between the internal

organs. This fat can compress the thin arteries that travel to the heart causing CHD. 6. Differentiate between Type I (red), Type IIa, and Type IIb (white) muscle fibers

a. Present the general structure of a muscle fiber including a description of the dystrophin protein- Dystrophin protein (Couldn’t find it in lecture)

Property Type I slow twitch Type IIA Fast oxidative

Type IIB Fast Glycolytic

ATP Hydrolysis Slow Fast FastContraction Speed Slow Fast FastGlycolytic Capacity Low Moderate HighOxidative Capacity High Moderate LowGlycogen Storage + ++ +++

Appearance Red Red WhiteCapillary Supply Good Moderate Poor

7. Compare energy metabolism in resting muscle and exercising muscle tissues

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a. Discuss fuel sources and relate to the fed/fast state- At rest muscle uses ~30% of oxygen. During vigorous exercise it uses ~90% of oxygen. In fed state in creased glucose transport via GLUT 4. During starving state you have an increase in FA uptake. In resting muscle high ATP levels drive the synthesis of creatine phosphate. In exercising muscle low ATP levels allow creatine phosphate to drive the synthesis of ATP from ADP.

b. Discuss the changes that occur during long duration exercise- Prolonged exercise of starvation the Blood glucose goes down, so that is a decrease in insulin and an increase in glucagon. This leads to an increase in FA usage. HSL is activated in adipose. Insulin dependence on glucose uptake may be overcome by elevated AMP levels. Signals through the AMP-protein kinase (AMPK) mobilizes GLUT 4 transporters to sarcolemma.

8. Differentiate aerobic and anaerobic skeletal muscle metabolism a. Discuss how creatine-phosphate functions as an ATP buffer- In resting muscle it

stores ATP because ATP is high energy and is used quickly, don’t want to waste it in a resting muscle. When muscle needs ATP in converts ADP ATP.

b. Discuss how fuel sources change in anaerobic muscle contraction- Lactate produced is used via the Cori cycle, muscle or heart. This happens when ATP requirements exceed aerobic capacity.

c. Discuss the end electron acceptor during energy generationi. Oxygen in aerobic exercise and lactic acid formation from pyruvate

ii. The central importance of oxidizing NADH to NAD+- Generates NAD+ for Ketone body synthesis. The Krebs cycle also needs NAD+

d. Review the regulation of glycogen degradatione. List the biological fuels that cardiac muscle may use under normal conditions-

Cardiac muscle may use any fuel. Always dependent on vascular supply. 9. Review the concept of the ‘blood brain barrier’ (BBB) in relation to brain metabolism

a. Fatty acids from TAG mobilization may not cross the BBB (dietary essential fatty acids may enter the brain)- Small uncharged molecules and non-polar substances freely pass.

b. Dietary essential amino acids may cross the BBBc. Explain the BBB metabolic block to L-DOPA – Amino acids that are NT are blocked

from entry to the brain to separate the somatic pools from the neural pools. This way if the brain needs a neurotransmitter it’ll just make it. Treatment of parkinsons is using L-DOPA. L-DOPA can cross the blood brain barrier but it will be quickly degraded by neuronal endothelial cells. DOPA decarboxylase inhibitor must be given in combination with L-DOPA for effective therapy.

10. Discuss the nutrient molecules that are used for energy production in the CNSa. The brain uses Glucose and this is not insulin responsive. Ketone bodies are used in

the brain and during starvation KB transporter in the brain is upregulated. 11. Compare the fed state to the changes that occur after prolonged fast

a. In the well fed state the diet supplies the energy requirements for the brain via glucose. In the fasting state, glycogenolysis and gluconeogenesis maintain glucose levels to supply brain with fuel. Under normal conditions there is abundant glucose

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in circulation. During a prolonged fast there is significant rise in serum ketone bodies. Ketone bodies become the major brain fuel

12. Identify the central role of glutamate in neurotransmission:a. Glutamate as a major excitatory neurotransmitter- Receptors found in post-

synaptic cell. Many subtypes named for drugs that bind the receptor. Glutamate signal termination occurs by reuptake in presynaptic terminals or by glial transporters.

b. GABA as a major inhibitory neurotransmitter- Epilepsy is associated with low GABA. Epilepsy may be treated with GABA analogs or drugs that block GABA reuptake from synapse.

c. Describe GABA metabolism in the brain, including the GABA shunt- GABA is recycled in the CNS GABA shunt. Conservation of glutamate and GABA. Glial cells contribute the major uptake of GABA. Glial cells lack the glutamate decarboxylase. GABA shunt converts GABA into TCA cycle intermediate so it can go back to α-ketoglutarate so it is essentially being recycled.

13. Describe the metabolism of acetylcholine in the braina. Dependence on SAM and vitamin B12- Required to regenerate SAM to form choline.

Needed for neurotransmitters (ACH). b. Action of acetylcholine esterase- Cleaves acetylcholine

14. Describe the structure and function of myelin and link it to the destruction of myelin that is observed in a patient with Multiple Sclerosis

a. Myelin is a multi layered membrane sheath made up of lipids and proteins with high content of sphingomyelin and cerebrosides (Speeds action potentials). Multiple sclerosis is characterized by a progressive destruction of CNS myelin. Formation of sclerotic plaques which slows neurotransmission (leads to eventual loss). An autoimmune reaction: possible triggered by a viral infection. Episodal periods of destruction and symptoms are weakness, lack of coordination, loss of vision.

15. Compare and contrast the biochemical mechanisms underlying the neurodegenerative disorders of Alzheimer’s and Prion diseases

a. Alzheimer’s is progressive loss of memory and cognition. Mitochondrial dysfunction in neural cells. Results from the inappropriate accumulation of proteolytic fragments from the β-amyloid precursor protein. The neurotoxic peptide has a high β-sheet content forms aggregates and neurofibrillary tangles.

b. Prion Disease- Changes in tertiary structure of the PrPc protein. Same thing as Transmissable Spongiform encephalopathy disease. Prions are proteins that may act as infectious agents when incorrectly folded. Normal prion protein (PRP) is soluble and is found on the extracellular side of neuronal membranes. The defective one has β-sheet and is resistant to protease digestion.

16. Revise the importance of vitamin B1 on brain metabolism (Reading assignment)a. Wernicke Korsakoff syndrome and Beri-beri- Moderatively severe thiamine

deficiency (B1), mental confusion, ataxia, opthalmoplegia. Most common in chronic alcoholics and responds to thiamine supplement.

17. Pathways of the biosynthesis of Catecholamines: dopamine, norepinephrine, epinephrine derivatives of tryptophan: Serotonin and melatonin (This is on other lectures)

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Lectures: Vitamins and Minerals I and II (2 lectures)

1. List and group the major vitamins of the human diet (water soluble and lipid soluble)a. Water Soluble- Vitamin C (Ascorbic Acid), Thiamine (B1), Riboflavin (B2), Niacin (B3),

Biotin, Pantothenic Acid, Folic Acid, Vitamin B12, Pyridoxine (B6), Pyridoxal, and Pyridoxamine

b. Fat-Soluble (Require Bile for Absorption)- Vitamin A, D, E, K (we do not store large amounts of vitamin K.

2. Discuss the significances of vitamin B and vitamin C deficiencies a. Vitamin C

i. Scurvy- fragile blood vessels, sore and spongy gums, bleeding into joints, impaired wound healing. Defects in connective tissue because of decreased hydroxylation of collagen. Less stable collagen. Can be caused by a lack of fruits and veggies in the diet.

3. Indicate the coenzyme forms, the reaction requiring and the biochemical basis of clinical manifestations of deficiency of thiamine (B1), riboflavin (B2), niacin (B3), and pyridoxine (B6)

a. Vitamin B1 (Thiamine)- Oxidative decarboxylation of alpha keto acids. Needed for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched chain α-ketoacid dehydrogenase. Acts as a coenzyme for transketolase in the HMP pathway. Polished rice, white flour and white sugar are deficient in TPP.

i. Beri Beri- When polished rice is major diet component. Disruption of motor, sensory and reflex arcs. Neurological manifestations is dry beri beri and cardiovascular symptoms is wet beri beri.

ii. Wernicke-Korsakoff Syndrome- Associated with chronic alcoholism. Ophthalmoplegia and nystagamus (to and fro movement of eyeballs).

b. Vitamin B2 (Riboflavin)- Forms FMN and FAD and participates in oxidation reduction reactions of TCA cycle, Beta oxidation. Succinate DH, Pyruvate DH ,and Acyl CoA DH (MCAD)

i. Cheilosis- areas of pallor cracks and fissures at the angles of the mouth. ii. Glossitis- inflammation of the tongue

iii. Facial Dermatitisc. Vitamin B3 (Niacin)- Coenzyme forms NAD+ and NADP+. Niacin inhibits lipolysis in

adipose tissue and greatly reduces production of free fatty acids. i. Pellagra- rough skin characterized by the 3 Ds (dermatitis, diarrhea and

dementia). Dermatitis is exposed areas of body, redness, thickening and roughening of skin. You will see people with necklace like skin damage and eventually leads to death. Tryptophan can be used to synthesize NAD+ and NADP+. Corn based diets can cause pellagra because it’s deficient in Niacin and Tryptophan.

d. Vitamin B6 (Pyridoxine)- Serve as precursors for PLP which acts as coenzyme for transamination, amino acid decarboxylation (dopa-decarboxylase, serotonin, and glutamine decarboxylase), ALA synthase and conversion of homocysteine to cysteine (Cystathionine β-Synthase).

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i. Deficiency leads to mycrocytic anemia (ALA synthase) and increased risk of cardiovascular disease because of increased levels of homocysteine.

ii. Isoniazid is an antituberculosis drug that inactivated pyridoxine. e. Vitamin B12 (Cobalamin)- Essential for synthesis of methionine and isomerization of

methylmalonyl CoA in odd number fatty acid degradation. In B12 deficiency folate is trapped as methytetrahydrofolate and results in macrocytic anemia. Lack of IF results in a poor absorption of B12 and pernicious anemia.

i. Significant amounts stored in the body and symptoms include macrocytic anemia. Folate is trapped and not available for purine/pyramidine synthesis. You get neuropsychiatric symptoms. Myeline degradation in both motor and sensory pathways due to methylmalonyl CoA accumulation. (If folate is deficient you will not get neurological symptoms).

1. Folate is acquired from the diet through green leafy vegetables. Can also be due to cobalamin deficiency (folate trap). Megaloblastic anemia has to be treated with folic acid and B12.

4. Describe the absorption of fat-soluble vitamins. Predict the reason for occurrence of fat soluble vitamin deficiency in patients with fat malabsorption

a. The absorption of fat soluble vitamins is dependent on normal fat digestion and absorption. Maldigestion and malabsorption of dietary fats results in secondary deficiency of the fat soluble vitamin (bile duct obstruction, cystic fibrosis). The fats aren’t being incorporated into chylomicrons so neither are the vitamins.

5. Describe the use of vitamin K as coenzyme for -carboxylation of inactive blood clotting factors in the liver.

a. Required post translational modification of various clotting factors (II, VII, IX and X). In humans it is also synthesized by bacterial flora. Vitamin K serves as a cofactor for liver microsomal γ-carboxylase. Vitamin K dependent γ-carboxylation of glutamic acid residues. The addition of another carboxyl group on respective glutamate residues leads to a renaming from Glu residues to Gla residues. γ-carboxylation allows formation of a mature clotting factor that is capable of subsequent activation. γ-carboxylation allows Calcium binding because of two adjacent negatively charged carboxylate groups. The clotting factor calcium complex can then bind to phospholipids on the platelet membrane.

6. Discuss hemorrhagic disease of the newborn. Predict the causes and effects of vitamin K deficiency.

a. The newborn has a sterile intestine and vitamin K2 is not formed by bacteria. Breast milk as natural nutrition for the newborn is low in vitamin K1. The enzymes for the synthesis of inactive blood clotting factors and for γ-carboxylation in the liver have still low activities in the newborn which will increase in the following days and weeks. Routine intramuscular injection of vitamin K is recommended for all newborns.

7. Discuss inhibition of -carboxylation by warfarin and compare and contrast the anticoagulant action of heparin and warfarin.

a. Warfarin interferes with γ-carboxylation of inactive blood clotting factors in the liver. It blocks the activity of liver epoxide reductase and prevents regeneration of

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reduced Vitamin K. This leads to a delay in clotting. These drugs do not have a short-term effect on blood clotting and are not used for prevention of blood clotting during surgery.

b. Heparin is the anticoagulant of choice to reduce blood clotting during surgery. Heparin facilitates the binding of anti-thrombin III to thrombin, and with that it limits the action of thrombin.

8. Describe the deficiencies of dietary vitamin A and retinoids and discuss use of retinoids as drugs in medicine

a. Collectively called retinoids. Dietary deficiency is the commonest cause – fat free diets and malabsorption of fats can also lead to deficiency. Signs and symptoms may include night blindness (Earliest), xerophthalmia (dryness of cornea), Bitot’s spots (white spots in cornea), Keratomalacia (softening/erosion of cornea), increased risk of pulmonary infections and weakened immune system.

b. Retinoic acid is used to treat severe acne and psoriasis. Too much can be toxic leading to Hypervitaminosis A (Headaches), dry and pruritic skin, enlarged liver, and spontaneous abortions in pregnancy. Vitamin A should be avoided in pregnancy.

9. Discuss the formation and action of retinoic acida. Retinol enters the target cell and is oxidized to retinoic acid in the cytosol. From the

cytosol, the retinoic acid moves into the nucleus with the help of cellular retinoid binding proteins. Retinoic acid binds to nuclear receptors forming an activated receptor complex. Retinoic acid-receptor complex binds to chromatin activating the transcription of specific genes (keratin).

10. Describe the function of cis-retinal in vision. Indicate why retinoic acid cannot be used to treat night blindness.

a. Retinol is transported to the retina and enters the retinal pigment cells. 11-cis retinol is oxidized to 11-cis retinal. 11-cis retinal enters the rod cell where it combines with opsin to form rhodopsin (visual pigment). Absorption of a photon of light catalyzes the isomerization of 11-cis-retinal to all-trans-retinal triggering a cascade of events, leading to the generation of an electrical signal to the optic nerve which is interpreted as vision.

b. Retinoic acid cannot be used to treat night blindness because it cannot be converted into retinol or retinal. The pigment cells need retinol/rods need retinal.

11. Describe the formation of vitamin D in the skin and the formation of calcitriol (role of liver and kidney) (Draw page 24 slide 19)

a. 7-dehydrocholesterol is converted to cholecalciferol by sunlight. This cholecalciferol goes to the liver where it is hydroxylated by 25-hydroxylase to 25-hydroxycholecalciferol. This molecule then goes to the kidney where it is hydroxylated again by 1-hydroxylase to Calcitriol. (1-hydroxylase is the RLS stimulated by parathormone and a decrease in calcium) 1,25 dihydroxycholecalciferol is the same thing has calcitriol.

12. Describe the functions of 1,25 dihydroxy-D related to calcium metabolisma. 1,25 Dihydroxycholecalciferol binds to intracellular receptor proteins. 1,25-

dihydroxy-D3 receptor complex interacts with DNA in the nucleus of target cells (intestine). They can then selectively stimulate gene expression or repress gene

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expression. It can stimulate the intestinal absorption of calcium and phosphate by increased synthesis of a specific calcium binding protein. On the bone it can stimulate the mobilization of calcium and phosphate form the bone in the presence of parathormone. On the kidneys can inhibit calcium excretion by stimulation parathyroid dependent calcium reabsorption. INCREASES PLASMA CALCIUM

13. Describe the biochemical basis of clinical manifestation in rickets and osteomalacia.a. Vitamin D deficiency in children and it is due to vitamin D deficiency in children. You

have a decreased calcium absorption in the diet increase in parathyroid hormone release increase in demineralization of the bone. Bones become soft and pliable and you get the characteristic bow leg deformity. You also get a pigeon chest deformity. Frontal bossing.

b. Osteomalacia is Vitamin D deficiency in adults. Bones are demineralized and are susceptible to fracture.

14. Discuss the role of copper as cofactor for enzymesa. Important cofactor in redox reactions. Cytochrome C is part of complex IV in the

ETC. Superoxide dismutase, lysyl oxidase (synthesis of collagen), and tyrosinase (neurotransmitter synthesis). Copper forms ceruloplasmin in the liver, which apart being a copper transport protein, helps in iron metabolism.

b. Ingested copper is absorbed in the stomach and intestine and transported to the liver via albumin. In the liver it is used to form ceruloplasmin which is secreted into the plasma. Aged ceruloplasmin is taken up by the liver from the plasma, endocytosed and degraded and copper is secreted into bile.

c. Signs and symptoms- microcytic anemia (smaller RBC) because iron metabolism needs copper. Degradation of vascular tissue because lysyl oxidase needs copper. Defects in hair.

15. Discuss clinical manifestations and biochemical defects of defective copper metabolism in Wilson’s disease and Menke’s syndrome.

a. Menke’s Syndrome (Kinky Hair syndrome)- Inherited defect in absorption of copper from GI tract. Low levels of copper in plasma and most tissues. Hair is twisty, grayish and “kinky.” Copper deficiency can lead to aneurysms and cerebral dysfunction.

b. Wilson’s Disease- Accumulation of toxic levels of copper in vital organs including liver, brain and eye. Defect is found in the copper transporting ATPase in the liver. This protein is needed to attach copper to ceruloplasmin and also to excrete copper into the bile. You see characteristic Kayser Fleischer Rings in the cornea. Decrease serum ceruloplasmin, increased urinary excretion of copper and increased hepatic copper content. (P 36 of objectives to look at ceruloplasmin)

16. Discuss the laboratory findings and biochemical basis for clinical manifestations in hereditary Hemochromatosis.

a. Required for heme synthesis and is important for redox reactions. The dietary iron absorption is tightly regulated by body iron stores. Higher the body iron stores less iron is absorbed by the intestine. Iron needs to be in ferrous state to be absorbed and the stomach converts ferric iron to ferrous iron. Ceruloplasmin (ferroxidase) participates in the release of ferrous iron from intestinal cells and forms ferric iron

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which is needed for transport in the blood. Transferrin is transport protein for ferric ion in blood plasma.

b. Most common nutritional deficiency. Hemochromatosis is excessive absorption of iron. HFE gene in intestine regulates iron uptake and it is defective in this disorder. Unregulated uptake of iron. More common in males and iron damages tissues by lipid peroxidation through free radicals and DNA damage. Also you see liver damage, diabetes (destruction of pancreas), cardiac dysfunction and brownish pigmentation of the skin.

17. Indicate the nutritional causes of microcytic anemia.a. Microcytic anemia is the result of reduced heme synthesis in erythroid cells. This can

be due to deficiency of pyridoxine which leads to deficiency of PLP needed as coenzyme for ALA synthase. [Glycine is decarboxylated in this step]. It can also result from deficiency of iron leading to less heme synthesis in erythroid cells. [Copper deficiency leads to iron deficiency]. Lead toxicity leads to microcytic anemia, as lead inhibits ALA dehydratase and ferrochelatase and less hemoglobin than before is formed in erythroid cells.

Lecture: Introduction to nutrition

1. Analyze the components of daily energy expenditurea. Resting energy requirement (REE/ RMR/ BMR) and enumerate factors affecting it

i. REE- resting energy expenditureii. RMR- Resting Metabolic Rate- can be effected by gender, body temperature,

environmental temperature, thyroid function, pregnancy and lactation and age.

iii. BMR- Basal metabolic rate- energy to support our metabolism b. Physical activity- Sedentary you have 30% expenditure of RMR, moderate activity

you have 60-70% expenditure of RMR, and several hours of heavy exercise you have 100% expenditure of RMR.

c. Diet induced thermogenesis (SDA)- Represents the energy required to digest, absorb, distribute and store nutrients.

2. Differentiate the macronutrient ratios of a balanced diet, energy content of macronutrients and list important sources of - carbohydrates, proteins, fats, ethanol and dietary fiber

a. Carbohydrates (45-65%)- Provide 4 kcal/g. Abundant in fruits, sweet corn, corn syrup and honey. Polysaccharides found in wheat, grains, potatoes, dried peas, beans, vegetables, starch and fiber (no energy but adds bulk to diet).

b. Fat (20-35%)- Provide 9 kcal/g. Corn oil, soybean oil, olive and canola oil, margarine. You want more saturated fats and increase unsaturated (ex- TAGs from plants).

c. Protein (10-35%)- Provide 4 kcal/g. Meat, poultry, milk, and fish. d. Ethanol- Provides 7 kcal/g.

3. Indicate the significance of dietary carbohydrate and predict the significance of glycemic index of carbohydrates

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a. Some carbs (high glycemic index) will raise serum glucose more rapidly than others and the decline in serum glucose will also be steeper. Needs carbs for glucose.

4. Determine the significance of dietary fats in relation to coronary heart disease, and essential fatty acids

a. Provide EFA linoleic and linolenic acids. EFAs required for membrane fluidity and synthesis of Eicosanoids. Deficiency of EFA characterized by scaly dermatitis, hair loss, and poor wound healing. CHD = blockage of heart.

5. Explore the significance of dietary fiber and correlate it to clinical applications of dietary fiber- Consists of non-digestible carbs. Included cellulose, lignin, and pectin. Adds bulk to diet and increases bowel motility. Decreases risk for constipation, hemorrhoids, diverticulosis and colon cancer.

6. Analyze the significance of dietary protein and differentiate between the sources of high and low quality protein. Indicate states of positive and negative nitrogen balance

a. Protein is needed to replenish body proteins. 20 are essential and the high value sources contain high amounts of EAA. Gelatin, wheat, corn, rice and beans are low biological value sources of protein.

b. Positive nitrogen balance is when N intake > N excretion (tissue growth)c. Negative nitrogen balance is when N excretion > N intake (inadequate dietary

protein, stress)7. Explore the roles of various hormones involved in appetite regulation (leptin, insulin,

ghrelin)a. Leptin- a peptide produced by adipose tissue. Leptin is produced proportionally to

fat cell density and plays a key role in weight gain, appetite suppression and energy expenditure through its action on the hypothalamus. It also regulates inflammatory responses, blood pressure and bone density

b. Insulin- similar to leptin, dampens appetite by exerting its effects on the hypothalamus.

c. Ghrelin- a peptide hormone secreted by the stomach that increases the release of neuropeptide Y. This enhances appetite and food intake. Ghrelin release is activated by fasting and low glucose and inhibited by high glucose. CCK and PYY cause satiety during a meal and transmit those signals to the hypothalamus.

8. Solve problems involving calculation of energy expenditure in an individual (based on activity)

9. Solve problems involving dietary macronutrient ratios for a balanced diet 10. Distinguish the methods to assess nutritional status (anthropometry and biochemical)

a. Anthropometry- BMI provides a measure of relative weight adjusted to height. Healthy range 18.5-24.9. Overweight 25-29.9. Obese is greater than 30. Mid-arm skin-fold thickness is used to assess subcutaneous fat by comparing it to population standards.

Lecture: Obesity

1. Definition of obesity in terms of BMIa. Obesity is when BMI = Weight (kg)/Height (m2) is greater than 30kg/m2

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2. Analyze methods for diagnosis of obesitya. Compare the various methods available for diagnosis of obesity: BMI ≥ 30, waist

circumference (≥ 40 for men, ≥ 35 female), Waist to hip ratio (≥ 0.9 for men, ≥ 0.8 for women), bioelectric impedance (measures conduction speed of a small electric current in the body)

b. Discuss the significance of location of body fat (‘apple’ vs ‘pear’) in obesity- apple is in the abdomen (visceral) and pear is in the thighs (subcutaneous).

3. Summarize the factors contributing (multifactorial) to obesitya. Genetic- Mutations in the leptin gene or its receptor can produce hyperphagia and

massive obesityb. Environmental and behavioral factors- Lifestyle and behaviorc. Explore the role of leptin in obesity- Previous lectures. Leptin is supposed to

suppress appetite. If you have a defect what happens?4. Analyze the metabolic changes in obesity

a. Dyslipidemia – Correlate biochemical changes in lipid profile to metabolic changes in obesity- Too much lipids in the blood so that leads to an increase in serum TAGs and VLDL and fat deposition.

b. Syndrome X (Metabolic syndrome) – Correlate clinical and biochemical features and explain the significance of insulin resistance- Extra weight around your waist leads to increased visceral fat and higher chance of CHD. You also have low levels of HDLs and insulin resistance. Results in increased TAGs in circulation due to increase synthesis of VLDL by the liver and results in increase risk of atherosclerosis. Insulin resistance is correlated with weight gain.

5. Explain the biochemical basis for management of obesitya. Decrease caloric intake and increase physical activity. Improve quality of food

consumed and increase intake of fruit and vegetable (increase fiber). 6. Determine the role of drugs (sibutramine and orlistat) and surgery in obesity

managementa. Sibutramine- an appetite suppressant (increases fullness- a feeling of satiety)b. Orlistat- gastric and pancreatic lipase inhibitor, inhibits the digestion of dietary fats

(TAGs) and therefore reduces dietary TAG absorption. c. Surgery- Recommended for morbidly obese BMI > 35. Decrease size of stomach, ileo

gastric bypass. They are used in conjunction with behavioral modifications to achieve long-term weight loss objectives.

Lecture: Starvation and Undernutrition

1. Summarize hormonal, metabolic changes and changes in the various organs (liver, muscle, brain, adipose tissue) in the various phases listed below

a. Postprandial phase (2-3 hours after a meal) – Glucose and insulin levels increase in blood after a meal

b. Post – absorptive phase (5-7 hours after a meal) - Usage of glucose leads to lower blood glucose levels. Insulin/glucagon ratio changes in favor of glucagon. Liver

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metabolism is characterized by release of glucose into the blood using glycogen degradation and gluconeogenesis. Release of FFA into the blood.

c. Early phase of starvation- Plasma levels of Fatty acids and ketone bodies increase while glucose decreases.

2. Prolonged starvation – adaptations to increase survival, role of kidney- Kidney performs gluconeogenesis.

3. Predict the deleterious effects of prolonged starvation- Substantial loss of body fat and muscle mass, vitamin and mineral deficiencies reduce enzyme activities, atrophy of intestine, diarrhea, skin rashes, edema, hypothermia and heart failure.

4. Correlate important clinical features with biochemical basis for anorexia nervosaa. Patients refuse to maintain normal body weight as they feel fat even when

undernourished. Related to low self esteem. They have low blood pressure, bad memory, fatigue, anemia, weak muscles, kidney failure, constipation and loss of menstrual cycle. Associated with Marasmus

5. Regarding protein energy malnutritiona. Distinguish and define types of PEM- Protein energy Malnutrition leads to variable

clinical conditions with extreme forms, including patients with major trauma, and depressed immune system.

b. Compare and contrast features of marasmus and kwashiorkor i. Marasmus- Chronic dietary restriction of carbs, lipids, proteins and other

nutrients. Extensive tissue and muscle wasting. Dry skin, loose skin folds hanging over buttocks. Drastic loss of body fat on buttocks and thighs. Associated with anorexia nervosa.

ii. Kwashiorkor- Deficiency of dietary protein and leads to muscle wasting due to lack of EAA. Decreased serum albumin so you have edema in abdomen and legs.

c. Correlate biochemical basis for the clinical features of both (above)d. Identify associated vitamin and mineral deficiencies in PEM- Vitamin deficiencies

can lead to beri-beri (B1), cheilosis (B2), pellagra (B3), scurvy (Vitamin C), anemia (B12), reduced blood clotting (Vitamin K), reduced response to infections and poor wound healing.

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Lecture: Metabolic response to trauma

1. Generalize the features of the hypermetabolic response and enumerate its causesa. Increase in glucagon, cortisol, ADH, Glucocorticoids, Insulin (However you have

insulin resistance). Increased catabolism of peripheral protein in skeletal muscle and lipolysis because of phagocytic release of mediators. Because of loss of fluid and electrolytes you have the release of catecholamines, aldosterone, ADH and glucocorticoids. Through Alanine-glucose cycle you use amino acids for gluconeogenesis and synthesis of acutely needed proteins like C-reactive protein.

2. Correlate hormonal and metabolic changes (carbohydrate, protein and lipid) following trauma in the three phases.

a. Ebb phase (Unresuscitated phase)i. Ebb phase- decreased cardiac output, O2 consumption, and body

temperature. This is the immediate response following an injury. You can have lactic acidosis due to decreased tissue oxygenation (decreased cardiac output). Insulin levels drop this is different from the flow phase. Carbs- you have low insulin levels with slight increase in glucose production.

b. Flow phase (Adrenergic – corticoid phase)i. Increased cardiac output, O2 consumption, body temperature, energy

expenditure and protein catabolism. Marked increase in glucose production, increase in free fatty acids because HS lipase is active because the body is not responding to insulin and catecholamines are activating it (No ketogenesis). You have an increase of catecholamines, insulin, glucagon, and cortisol. Amino acids are used for the synthesis of acute phase proteins. Carbs- you have high blood glucose, with high insulin (resistance), decrease uptake of glucose, increase in gluconeogenesis. Glucose is used by the injured tissue and converted to lactate leading to lactic acidosis. Protein- Negative nitrogen balance and skeletal muscle is the major source of the N2

loss following injury. Glutamine and Alanine are majority of the released Amino acids. They are used for gluconeogenesis or acute phase protein synthesis.

c. Recovery phase (convalescent/ anabolic phase)- Not in lecture3. Indicate the significance of acute phase proteins in response to trauma

a. Measurement of acute-phase proteins, especially C-reactive protein, is a useful marker of inflammation

4. Explore the significance of nutritional support and role of glutamine supplementationa. Glutamine is given for enhancement of immune function. Supplementation is

reported to improve immune functions (this is everything that’s on the slide about this)

5. Distinguish the pros and cons of enteral vs parenteral nutrition supporta. Enteral Nutrition- via the gut and preserves intestinal mucosal integrity better than

parenteral nutrition with bowel rest. You have decrease in mortality, bacterial

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translocation to mesenteric lymph nodes, decrease in sepsis, and it preserves GI flora better. A prerequisite is that you have a functional GI tract.

b. Parenteral nutrition is not as good and it is through IV and it bypasses the gut. This is not good because it doesn’t preserve the intestinal mucosal integrity.

6. Distinguish the metabolic response to stress and starvationSimple Starvation Severe Injury

BMR Inflammatory Mediators - +++

Major Fuel Fat Protein + FatKetone Bodies +++ -

Negative N2 balance + +++Blood Glucose Level Lower

Gluconeogenesis and Proteolysis

+ +++

Hepatic Protein Synthesis + +++ (C-Reactive Protein)

Lecture: Diabetes mellitus

1. Define diabetes mellitusa. Group of disorders characterized by the presence of hyperglycemia that result from

defects in secretion of insulin OR action of insulin OR both2. Distinguish between the types of diabetes: type I and type II (differences between the two

types in terms of insulin) a. Type I diabetes is insulin dependent diabetes and it is autoimmune destruction of

the β-cells of the pancreas and marked decrease in insulin secretion. Patients have to be on life long insulin supplementation to prevent complications of diabetes.

b. Type II is non-insulin dependent and Obesity (syndrome X) is an important risk factor for development of type II diabetes mellitus and insulin resistance. Target tissues for insulin do not respond to circulating insulin and there is a decrease in insulin secretion with time.

i. In the early stages there is a hypersecretion of insulin and target tissues are not responding to insulin. In the later stages there is decreased secretion of insulin from the β-cells (β-cell fatigue) combined with insulin resistance.

3. Correlate the presenting features of diabetes and biochemical basis for the clinical features

a. Polyuria- You pee more because you have a lot of glucose. Not all the glucose is reabsorbed in the kidney so when you pee the glucose takes water with it.

b. Polydipsia- Since you are peeing more and the glucose in the pee is taking water you feel more thirsty

c. Polyphagia- You are hungry because you have insulin resistance and leptin resistance is usually coupled with this (I think this is right)

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4. Summarize the complications of diabetes and correlate to biochemical and metabolic changes

a. Acute complicationsi. Ketoacidosis (Type I)- Uncontrolled lipolysis of adipose tissue because of

no insulin. Results in increased FFA to the liver and increased β-oxidation. The results in the formation of acetyl CoA for ketone body synthesis. Ketogenesis >>> Peripheral utilization of ketone bodies. Ketone bodies (Weak acids) in blood. You get a fruity odor because some ketone bodies spontaneously get converted to acetone.

ii. Hyperosmolar non-ketotic coma- Coma is due to Hyperglycemia and because glucose is being excreted it is taking water with it. This causes neuronal dehydration.

b. Chronici. Microvascular- Neuropathy, nephropathy and retinopathy are

microvascular complications. These are tissues which do not require insulin for glucose entry (retina, nervous tissue, lens). Sorbitol formation in the lens, nerve and kidney results in cell swelling due to water retention. Glycation of proteins (AGEs) of the basement membrane contributes to nephropathy. You also get foot ulcers and increased excretion of albumin in urine in the initial stages. **AGE is advanced glycation of proteins.

ii. Macrovascular- You have hyper TAGs. There is increased chylomicrons and VLDL in the circulation. This is due to decreased action of lipoprotein lipase present in the endothelium of blood vessels (LPL needs insulin for optimal activity). Insulin resistance increases the risk of cardiovascular disease in diabetes.

5. Acute complications of diabetes mellitus: a. Sequence of metabolic changes resulting in diabetic ketoacidosis (type I)- You need

insulin to stop HS lipase from releasing FFA and the liver from converting them to ketone bodies. Because type I has no insulin you cannot regulate any of these processes resulting in an increase in ketone bodies causing metabolic acidosis.

b. Compare and contrast biochemical and laboratory findings in hyperosmolar non-ketotic coma and diabetic ketoacidosis- Hyperosmolar is found in Type II while diabetic ketoacidosis is found in Type I. Ketosis is not prominent as in type I diabetics because you have insulin that can regulate HS lipase.

c. Relate the occurrence of hypoglycemia in diabetic patients on insulin- Sometimes you can give them too much insulin and that results in a depletion of serum glucose.

6. Evaluate the significance of the laboratory tests for diabetesa. Tests for diagnosis of diabetes: Fasting blood glucose, postprandial blood glucose,

role of GTTi. Fasting blood glucose > 126 mg/dL

ii. Elevated postprandial blood glucose (blood glucose obtained 2 hours after a meal)

iii. Oral glucose tolerance tests is performed on an individual after an overnight fast. Subject is given a known load of glucose orally. Blood

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glucose is estimated at the beginning of the test and every half hour for up to 2 hours. In a normal person the 2 hr blood glucose is less than 200 mg/dL. Diabetic patient has decreased tolerance to glucose.

b. Tests for long term management: i. Tests to assess glycemic control (Fasting blood glucose, postprandial blood

glucose, glycated hemoglobin) ii. Tests for lipid profile (Fasting lipid profile, serum cholesterol, serum TAGs,

LDL levels, HDL levels)iii. Tests to assess renal function- (measurement of albumin excretion in the

urine (microalbuminuria)

Lecture: Hypoglycemia

1. Summarize the hormonal regulation of blood glucosea. At low blood glucose levels several systems are used to normalize blood glucose. The

pituitary gland and ACTH, the autonomic nervous system or directly via the low serum blood glucose levels acting on the α-cells of the pancreas

2. Analyze the effects of hypoglycemiaa. Explain the release of counter-regulatory hormones

i. Response to low blood glucose levels leads to a release of catecholamines and cortisol. The cortisol promotes gluconeogenesis while the norepinephrine and epinephrine stimulate glycogenolysis. Glucagon from the α-cells of the pancreas stimulates both glycogenolysis and gluconeogenesis.

b. Discriminate the manifestations of hypoglycemia - Adrenergic, neuroglycopenic symptoms

i. Hypoglycemia is characterized by a blood glucose level less than 40mg/dL. Severe hypoglycemia is characterized by blood glucose less than 20 mg/dL. Adrenergic symptoms start at glucose blood levels of about 55 mg/dL and they include anxiety palpitations, tremor and sweating. Neuroglycopenia symptoms start at glucose blood levels at about 50 mg/dL and they include headache, confusion, slurred speech, seizures and even coma and death

ii. The types of hypoglycemia include insulin-induced hypoglycemia in patients treated with insulin or in patients with insulinoma. Postprandial hypoglycemia due to exaggerated insulin release in some individuals. Fasting hypoglycemia signals a serious medical problem, often genetic defects. Lastly you have hypoglycemia due to alcohol intoxication (High NADH ratio consumes gluconeogenesis intermediates can’t form OAA from malate).

3. Distinguish and discuss the mechanisms of causation of hypoglycemia and the differences between them

a. Fasting hypoglycemia- found at high ethanol levels and especially in undernourished fasted or dehydrated individuals that consume ethanol (high NADH ratio interferes with usage of gluconeogenesis precursors). Lactic acidosis also occurs because pyruvate lactate and leads to gout as uric acid and lactate build up in kidney. Also found in genetic defects of beta oxidation (MCAD deficiency, carnitine shuttle

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deficiency and CPT I deficiency). CPT II defect does not cause hypoglycemia because it is found in muscle and muscle cells cannot do gluconeogenesis. Lastly you can find hypoglycemia in Von Gierke’s disease (very severe) because you don’t have glucose 6-phosphatase so glucose cannot be released from the cell.

b. Reactive (postprandial) hypoglycemia- Postprandial hypoglycemia is quite common and occurs in individuals with exaggerated insulin release after a meal. This is not a severe condition and leads to transient hypoglycemia with mild adrenergic symptoms. Plasma glucose levels return to normal without treatment and preventable by frequent small meals

c. Alcohol induced hypoglycemia- Ethanol-induced hypoglycemia is found in individuals after excessive intake of ethanol. This is often found in undernourished, fasted or dehydrated individuals. High levels of ethanol in liver cytosol lead to fasting hypoglycemia when the glycogen stores are depleted and gluconeogenesis is the source for release of glucose into the blood. The high cytosolic NADH/NAD+ ratio prevents the normal usage of lactate, amino acids and glycerol for gluconeogenesis. NADH is formed in cytosol by alcohol dehydrogenase and also by cytosolic acetaldehyde dehydrogenase.

d. Factitious hypoglycemia- (Needs to be investigated) It is a mental/personal disease where the patient tries to acquire hypoglycemia due to injection of insulin or usage of specific medical drugs. You have to determine serum insulin, C-peptide, proinsulin and sulfonylurea. If only insulin is high and C-peptide and proinsulin are not elevated, then insulin was injected. If insulin, C-peptide and proinsulin are high then endogenous overproduction of insulin took place. Sulfonylura is an antidiabetic drug used for treatment of diabetes mellitus Type 2. This drug stimulates insulin secretion.

e. Hypoglycemia of insulinoma- This type of hypoglycemia is grouped as insulin-induced hypoglycemia. Severe hypoglycemia can occur during fasting in patients with insulinoma. The tumor produces high levels of insulin which blocks the action of the insulin counter-acting hormones. Patients with insulinoma have high blood glucose levels of insulin, C-peptide and proinsulin (Endogenous).

4. Justify the significance of estimation of serum insulin, C-peptide, proinsulin in the various types of hypoglycemia

5. Integrate knowledge from previously studied metabolic pathways to identify some of the causes of hypoglycemia in infancy and childhood (von Gierke’s disease, MCAD and carnitine deficiency) Kind of described above…this is in Trotz questions but both all of them cause hypoglycemia. MCAD and Carnitine cause hypoketonemia in addition to hypoglycemia.

Lecture: Molecular mechanisms in inherited diseases

1. Describe the molecular and biochemical basis for symptoms, diagnostic tests and treatment of

2. Cystic Fibrosis

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a. Autosomal recessive disorder in Caucasian children. Due to a mutation in the gene for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a membrane protein that acts as a chloride channel. Belongs to the group of an ATP binding cassette transporter. Phosphorylation of the cytoplasmic regulatory domain by PKA activates the channel providing regulated Cl- and fluid secretion. In the ducts it traps the Cl- in the cell creating a more viscous mucosa. In the sweat glands the CFTR is involved in the reabsorption of NaCl. Very little NaCl is reabsorbed resulting in a high salt content in sweat.

b. Symptoms associated with CF include malabsorption, abnormal sweat electrolytes, chronic pancreatitis, lung abscess, chronic bronchitis and honeycomb lung.

c. The mutant CFTR doesn’t transport Cl- into the airway lumen and as a result Na and H2O content of the luminal secretions is low resulting in thickened and viscid mucus secretions. More prone for bacterial infections. Respiratory infections are the most common cause of mortality and morbidity in patients with cystic fibrosis.

d. Exocrine Pancreas deficiency because loss of CFTR leads to thicker acinar secretions within the duct lumen leading to obstruction and tissue destruction. Fibrotic tissue and fat replace the pancreatic parenchyma – hence the name cystic fibrosis. Leads to maldigestion of nutrients and to excretion of fat in the stool (steatorrhea). More than 95% of males with CF are infertile because they lack a vas deferens a phenotype known as congenital bilateral absence of the vas deferens (CBAVD) .

e. Genetic Basis- Results from mutations at the single gene locus on the long arm of chromosome 7. Most common CF disorder is of ΔF508. It is a 3-bp deletion that eliminates the phenylalanine residue of CFTR at position 508. ASO test is useful if the mutation is known,

3. Sickle cell anemiaa. Point mutation in the β-globin chain of hemoglobin. Glutamic acid is replaced by

valine at the sixth position of hemoglobin. Replacement of valine in sickle cell disease hemoglobin tends to aggregate to form long filament like structures. This aggregation results in distortion of the structure of RBC-sickling.

b. The sickled and distorted cells are periodically removed by the spleen hence the patients have anemia. Spleen may also be enlarged (Splenomegaly). Since RBCs are being destroyed you get anemia and pre-hepatic jaundice. Characterized by high levels of unconjugated bilirubin. Urine is normal color and due to the excessive loss of conjugated bilirubin in the bile people may develop pigmented gall stones.

c. Biochemical diagnosis- Hemoglobin electrophoresis: HbS (Valine) has fewer number of negative charges than HbA (Glutamate). HbS moves slower than HbA toward the anode (+). ASO test (Dot Blot test) using this assay it is possible to identify heterozygous carriers.

4. Duchenne Muscular Dystrophy and Becker Muscular Dystrophy- a. Both the disorders are due to mutations in the dystrophin gene. They dystrophin

gene is the largest gene located on the X-chromosome. DMD is due to the almost complete absence of functional dystrophin. BMD is due to the production of abnormal dystrophin or less amounts of dystrophin.

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b. Duchenne Muscular Dystrophy- due to mutations in a gene on the X chromosome that prevent the production of dystrophin, a muscle protein. X-linked recessive disease and more common in males. Most patients die in their early 20s due to breathing and heart problems.

c. Becker Muscular Dystrophy- BMD is a milder form DMD. Symptoms are similar to the DMD but the onset is later and the course of the disease is milder. Both DMD and BMD result from a mutation in the dystrophin gene. The DMD deletions can however be distinguished from deletions causing BMD, when viewed at the codon level. Almost all deletions causing DMD involve frameshift mutations, abolishing translation of dystrophin protein. Many of the mutations also involve large deletion of exons. BMD deletions are in frame hence some dystrophin although truncated is translated.

5. Identify the differences between Duchenne muscular dystrophy and Becker muscular dystrophy with regard to severity of the disorder, type of mutations, alterations in dystrophin

a. DMD- Because of weakened muscles they have a hard time from rising from the floor Gower’s maneuver. Dystrophin is encoded by the largest gene described to date. Gene expressed mainly in smooth, cardiac, and skeletal muscle, with lower levels in the brain. Dystrophin anchors the cytoskeleton of muscle cells to the EC matrix. Dystrophin links actin filaments to transmembrane proteins of the muscle cell plasma membrane. Functional loss of dystrophin leads to oxidative cellular injury and myonecrosis.

b. Western blot has smaller protein size, and reduced quantity of dystrophin protein in BMD. Complete absence of dystrophin in DMD. Reduced quantity of complete absence of dystrophin in DMD. Serum Creatine kinase (MM), levels are elevated in patients with muscular dystrophy (indicative of muscle damage). Females are carriers and they usually have higher levels of CK-MM in serum. Multiplex PCR analysis is usually used to identify the specific mutation.

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