BIOCHEMISTRY LABORATORY: PROBLEM BASED LEARNING EXERCISESAmino Acids and Proteins

Answers to Questions:

CASE 11. Explain the biochemical basis for the patient’s disease.

Alzheimer’s disease is a progressive neurodegenerative brain disease wherein the component of the amyloid plaque that accumulates is amyloid β (Aβ), an extracellular peptide. This peptide, when aggregated in a β-pleated sheet configuration, is neurotoxic, and is the central pathogenic event leading to the cognitive impairment characteristic of the disease.

The Aβ that is deposited in the brain in AD is derived by proteolytic cleavages from the larger amyloid precursor protein. The proteolytic cleavage is achieved by the action of the enzyme secretase. The Aβ peptides aggregate, generating the amyloid that is found in the brain parenchyma and around blood vessels.

2. Explain the theories involved in the development of Alzheimer disease.a. Formation and accumulation of amyloid plaques (amyloid hypothesis)

Amyloid β is a peptide produced by enzymatic cleavage from amyloid precursor protein. This amyloid β, when aggregated in a β-pleated sheet configuration, forms amyloid plaques which is neurotoxic and causes cognitive impairment.

b. Accumulation of neurofibrillary tangles inside neurons (tau hypothesis)A second biologic factor involved in the development of Alzheimer disease is the accumulation

of neurofibrillary tangles inside neurons. A key component of these tangled fibers is an abnormal form (hyperphosphorylated and insoluble) of the tau (τ) protein, which, in its healthy version, helps in the assembly of the microtubular structure. The defective τ appears to block the actions of its normal counterpart.

CASE 2

Clinical impression: SCURVY1. Explain the biochemical basis for the patient’s disease.

Collagen contains hydroxyproline (hyp) and hydroxylysine (hyl) which are not present in most other proteins. These residues result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains. These hydroxylation reactions require molecular oxygen, Fe+2, and the reducing agent vitamin C (ascorbic acid). If there is deficiency in ascorbic acid, the hydroxylating enzymes (prolyl hydroxylase and lysyl hydroxylase) are unable to function leading to lack of prolyl and lysyl hydroxylation. Moreover, interchain H-bond formation is impaired, as is formation of stable triple helix in collagen.

2. Explain the biochemical basis for the patient’s signs and symptoms.

Scurvy is a disease characterized by sore and spongy gums, loose teeth, fragile blood vessels, and poor wound healing. Hydroxylation of proline and lysine is important in the synthesis of collagen. Hydroxyproline is important in stabilizing the triple helical structure of collagen because it maximizes interchain hydrogen bond formation. A deficiency in vitamin C leads to lack of hydroxylation of the two amino acids which now leads to impairment of H-bond formation and unstable triple helix of the collagen. Additionally, collagen fibrils cannot be cross-linked, greatly decreasing the tensile strength of the assembled fiber. With this, the connective tissue then becomes defective, resulting to the above signs and symptoms that characterizes scurvy. Fragile capillary (fragile blood vessels) can cause subcutaneous extravasation of blood, which explains the multiple hemorrhage present near the distal end of the patient’s nail.

***In scurvy, which is due to vitamin C deficiency, hydroxylation of proline residues is decreased, and an unstable form of collagen is produced. Bones, teeth, blood vessels, and other structures rich in collagen de-velop abnormally. Bleeding gums and poor wound healing are often observed.

Reference:

Harvey, R. & Ferrier, D. (2011). Lippincott’s Illustrated Reviews: Biochemistry 5th edition. pp. 20-21, 45-47, 377

Koeppen, B.M. and Stanton, B.A. (2010). Berne and Levy Physiology 6th edition. Mosby, Inc. (Elsevier): Canada page 38

CASE 3

1. Explain the difference between human insulin and Humalog mix 75/25 as to structure and phar-macokinetics.

Chemical Structure: Insulin contains 51 amino acids arranged in 2 polypeptide chains, Chain A and Chain B.

Chain A is made up of 21 amino acids, while Chain B consists of 30 amino acids. Residues A7 to B7 and A20 to B19 of chains A and B are covalently bonded by disulfide bridges.

Structure of human proinsulin (C-peptide plus A and B chains) and insulin. Insulin is shown as the shaded (orange color) peptide chains, A and B. In insulin lispro, the proline and lysine, at B28 and B29 respec-tively, have been reversed.

Insulin lispro, the first monomeric insulin analog to be marketed, is produced by recom-binant technology wherein two amino acids near the carboxyl terminal of the B chain have been reversed in position: Proline at position B28 has been moved to B29, and ly-sine at position B29 has been moved to B28.

The Humalog mix 75/25 is a mixture of insulin lispro solution, a rapid-acting blood glucose-lowering agent and insulin lispro protamine suspension, an intermediate-acting blood glucose-lowering agent.

Pharmacokinetics:

If the 2 amino acids in insulin lispro are reversed it would not have any effect with its half-life, its binding to the insulin receptor or its immunogenicity, which are similar to those of human regular insulin. On the other hand absorption for the Humalog mix is faster than regular insulin. Insulin lispro begins to exert its effects within 15 minutes of administration. Human insulin has a slower onset of action thus; it should be injected 30 minutes to an hour before meals. Peak serum concentrations of Humalog 75/25 occur 90 minutes after subcutaneous administration. With subcutaneously administered human in-sulin, peak serum concentrations occur within two to three hours.

2. Explain the biochemical basis for the difference in onset of action between insulin and Humalog mix 25/75.

Reversal of the proline at B28 and the lysine at B29 results in a more rapid dissolution in insulin lispro to a dimer then to a monomer that is more rapidly absorbed through subcutaneous injec-tion.

The very low propensity of 75/25 Humalog serves as its advantage in contrast to human insulin, to self-associate in an antiparallel structure and form dimers. Lispro dissociates into monomers almost instantaneously after administration through injection. This characteristic causes a more rapid absorption as compared with regular insulin giving a shorter onset of action.

REFERENCE: Katzung, B. (2012). Basic & Clinical Pharmacology. 12th ed. New York: McGraw-Hill Medical p. 744 Brunton, L. (2011). Goodman & Gilman's The Pharmacological Basis of Therapeutics 12th ed. Mc-

Graw-Hill Professional. pp. 1250-1251)

ONSET PEAK DURATIONa. Human regular insulin 30 min – 1 hour 2 – 3 hours 4 – 6 hours

b. 75/25 Humalog

(75% Protamine lispro/25% Lispro)

Less than 15 min 1.5 hours Up to 10 – 16 hours

CASE 4: 1. What is the most likely diagnosis? Justify your answer.

Based on the description of the pain that she’s experiencing, the patient most likely has sickle cell disease; which is common among the people with African-American descent.

The pain that the patient is experiencing is due to anoxia because the sickle cells block the flow of blood into the capillaries, thus, there is interruption in the oxygen supply which explains her fatigue and the paleness of her mucosal membranes. Her condition was triggered by the urinary tract infection she is currently experiencing (based on the results of her urinalysis, and white blood cell count, frequent and burning urination)

2. Explain the biochemical basis for the patient’s condition.Single amino acid substitution on the hemoglobin β chain. A molecule of HbS contains 2 normal

α-globin chains and 2 mutant β-globin chains, in which glutamate at the sixth position has been replaced with valine.

*** Hemoglobinopathies result from mutations that produce alterations in the structure of hemoglobin. There are many described hemoglobinopathies. One common mutation results in sickle cell anemia, in which the β chain of haemoglobin contains a valine rather than a glutamate at position 6 (designated as E6V, using the sin-gle-letter codes for the amino acids. E6V means that the glutamate [E] at position 6 in the amino acid chain [where the amino terminal is amino acid number 1] has been replaced by a valine [V]). Thus, in the mutant he-moglobin (HbS), a hydrophobic amino acid replaces an amino acid with a negative charge. This change allows deoxygenated molecules of HbS to polymerize. Red blood cells that contain large complexes of HbS molecules can assume a sickle shape. These cells undergo hemolysis, and anemia results. Painful vaso-occlusive crises also occur, and end-organ damage may result.

References: Denise R. Ferrier, Lippincott's Illustrated Reviews Biochemistry, 6th edition, pages 35-37