TYPE I DIABETES MELLITUS CASE: Walley Waterhouse, a 13 year old Caucasian boy was brought to the emergency room by his older sister who was 18 years old. The parents were out of town. The patient was nauseated, listless, and had polyurea and polydipsia. He had the flu two or three weeks earlier, but seemed to recover before his parents left for a vacation. The patient was 61 inches tall and weighed 93 pounds. His sister said that he had lost more than 5 or 10 pounds over the last month. His temperature was 101 degrees Fahrenheit, pulse was 99 beats per minute, respiration 28 breaths per minute (12 is normal). You start an intravenous infusion of normal saline and order laboratory tests: LABORATORY RESULTS FROM SERUM (S), BLOOD (B) and URINE (U) - Walley's VALUES REFERENCE RANGE - Wallie's VALUES REFERENCE RANGE Sodium (S) 143 mmol/L 136 - 145 mmol/L Creatinine (S) 0.8 mg/dL 0.3 - 0.7 mg/dL Potassium (S) 5.0 mmol/L 3.8 - 5.1 mmol/L Hemoglobin (B) 14 g/dL 11 - 16 g/dL Chloride (S) 102 mmol/L 98 - 108 mmol/L Leukocyte count (B) 16 X 103/uL 4.5 - 11 X 103/ uL CO2 (S) 14 mmol/L 23 - 30 mmol/L  Albumin (S)  4.6 g/dL 3 .7 - 5.4 g/dL  Glucose (S) 290 mg/dL 75 - 105 mg/dL  Protein  7.0 g/dL 6.3 - 8.1 g/dL  Specific gravity (U)  1.025  1.003 - 1.030 Ketone Bodies (U)  moderate  negative Glucose (U)   3+  Scale is 0 to 5+, 0 is normal Protein, Heme, Nitrite (U)   negative  negative  Urea nitrogen  19 mg/dL  7-19 mg/dL       Because you suspected metabolic ketoacidosis secondary to diabetes (diabetic ketoacidosis), you ordered the following tests: LABORATORY RESULTS FROM SERUM (S) AND ARTERIAL BLOOD (Ba) - Walley's VALUES REFERENCE RANGE - Wallie's VALUES REFERENCE RANGE pH (Ba) 7.30 7.35 -7.45 Ketones (S) moderate* - pCO2 (Ba) 31 mm Hg 35 - 45 mm Hg pO2 (Ba) 108 mm Hg 75 - 100 mm Hg Oxygen Saturation (Ba) 98% 95- 98 % - - - * measured using nitroprusside reaction. Your diagnosis was diabetic ketoacidosis. You placed the patient on intravenous saline containing insulin. You monitored the patients progress with periodic laboratory tests: serum glucose, bicarbonate, sodium, potassium, chloride, phosphate, urea nitrogen, and Creatinine levels. The patient was started on twice daily subcutaneous injections of intermediate-acting and regular human insulin. Before discharge the patient was taught to inject insulin and to monitor blood glucose and urine ketones.

EXPLANATION FOR OBJECTIVE I: Your first reference can be any hardcopy reference or text except your assigned text. Your second reference must come from a web source. Don't forget that there are specialized reference texts on diabetes in the library. The objective is to understand how the carbonic acid/bicarbonate buffering system responds to uncontrolled type 1 diabetes. IA. The laboratory test for serum CO2 is called the total CO2 or plasma carbon dioxide content. It is derived by adding a strong acid to a sample of plasma and measuring the CO2 released. The value for the plasma carbon dioxide content is often used as an estimate of the HCO3- content. Write the reactions for the conversion of protons and bicarbonate into carbonic acid and then into carbon dioxide gas. Also, look up the values for dissolved [HCO3-],[CO2(g)] and [H2CO3] in blood. Then use the equations and the concentrations to explain why the plasma carbon dioxide content is a fair approximation for the bicarbonate content in plasma. Answer: As shown below, the bicarbonate content accounts for almost all (95%) of the carbon dioxide content of plasma. Therefore, the plasma carbon dioxide content is a rough estimate of plasma bicarbonate. References: http://www.mdconsult.com; Ravel: Clinical Laboratory Medicine, 6th ed.; Mosby - Year book, Inc. 1995; 394-395 Guyton & Hall: Human Physiology & Mechanisms of Disease; W.B. Saunders Company. 1997; pg 257 and 263 IB. By looking at the first set of laboratory tests, you suspected metabolic acidosis and perhaps, diabetic ketoacidosis. Which tests tipped you off? Answer: For metabolic acidosis it was the low plasma CO2 and low pH. Low plasma CO2 is caused by either respiratory alkalosis or metabolic acidosis but the pH only drops with metabolic acidosis. Because of the glucosurea, hyperglycemia and ketonurea, you suspect diabetic ketoacidosis. References: http://www.harrisonsonline.com; Chapter 50 Acidosis and Alkalosis; http://www.mdconsult.com.; Goldman: Cecil Textbook of Medicine, 21st ed. W.B. Saunders Company; 2000; pg 560 Guyton & Hall: Human Physiology & Mechanisms of Disease; W.B. Saunders Company. 1997; pg 256 and 263 IC. Above, you wrote the reactions for the interconversion of bicarbonate, protons, carbonic acid and carbon dioxide gas. Use the equations to explain what would happen to the concentration of these compounds in Walley's blood if your guess of metabolic acidosis is correct but the respiration rate did not increase, i.e., that respiratory compensation did not happen. Answer: If H+ is increased by the addition of acid from ketone bodies, then the reaction would be pushed to the left and bicarbonate would be consumed. Assuming a constant rate of respiration, the concentration of carbonic acid and carbon dioxide gas should remain constant (or increased by a negligible amount). Follow up Question: The serum CO2 decreased by about 10 mmol/L. What was the increase in dissolved CO2? Answer: No significant increase because the pressure of CO2 in the lung will determine the dissolved CO2. References: Ganong, William F., Review of Medical Physiology. 19th ed., Lange Medical Books, 1997; pg 701 www.mdconsult.com; Goldman: Cecil Textbook of Medicine, 21st ed. 2000 ID. Respiratory compensation for acute metabolic acidosis begins rapidly. Hyperventilation is initiated in a few minutes by the rise in hydrogen ions in the blood. Chemoreceptors in the central nervous system are involved in the stimulation of respiration. Use the Henderson Hasselbach equation to explain the effect that increased respiration has on the pH of blood. Answer:  Increased respiration decreases the PCO2. At the same time, the concentration of bicarbonate is only slightly changed by the decrease in PCO2. Since PCO2 is the denominator in the Henderson Hasselbach equation, a decrease in PCO2 results in an increase in the pH. References: Ganong, William F., Review of Medical Physiology. 19th ed., Lange Medical Books, 1997; pg 699 www.mdconsult.com; Goldman: Cecil Textbook of Medicine, 21st ed. 2000; pg 558 http://www.harrisonsonline.com; Chapter 50 Acidosis and Alkalosis; IE. Demonstrate the effect that respiratory compensation had in maintaining Walley's blood pH. First, calculate the pH of Walley's blood if acute metabolic acidosis had occurred without respiratory compensation. Use the Henderson Hasselbach equation. Next, calculate the pH of Walley's blood after respiratory compensation. Assume that Walley's blood bicarbonate was originally 25 mM and that his normal blood PCO2 was 40 mm Hg before respiratory compensation. To determine the pH after respiratory compensation, assume that, on the average, the respiratory compensation for a 1 mM reduction in bicarbonate leads to an approximately 1.1 mm Hg ( 0.033 mM ) reduction in the partial pressure of carbon dioxide. Answer: Without compensation: According to the plasma carbon dioxide content, Walley's bicarbonate was reduced to about 14 mM. His PCO2 was 40 mm Hg or 1.2 mM. See the second equation. With compensation: Walley's had a bicarbonate reduction of about 24 -14 = 10 mM. This would correlate with about a 11 X 0.033 = 0.363 mM drop in carbonic acid. The final carbonic acid concentration was 1.2-.363 = 0.84. See the third equation.   References: Ganong, William F., Review of Medical Physiology. 19th ed., Lange Medical Books, 1997; pg 699 www.mdconsult.com; Goldman: Cecil Textbook of Medicine, 21st ed. 2000; pg 558 http://www.harrisonsonline.com; Chapter 50 Acidosis and Alkalosis;

EXPLANATION FOR OBJECTIVE II: Demonstrate that you understand what insulin is and how insulin functions by answering the following objectives: IIA. Draw a cartoon representation of a human insulin molecule. Label the chains, the N-terminus of each chain, the C-terminus of each chain, and indicate the number of amino acids in each chain. Show and label any bonds between sulfer atoms on cysteine.  Answer: References: Porte, Daniel, M.D., Sherwin, Robert, M.D., Ellenberg & Rifkin's Diabetes Mellitus, 5th ed. Appleton & Lange 1997; pg 30 Kahn, Ronald, M.D., Weir, Gordon, M.D., Joslin's Diabetes Mellitus; 13th ed., Williams & Wilkins; 1994; pg 39 http://www.accessscience.com (Key words: insulin structure) IIB. In what form is insulin just before it is secreted from the cell? Is it in solution or is it in a crystalline precipitate? Is it a monomer, dimer, hexamer.....,etc.? Is it complexed with a metal ion? Answer: At the time of fusion of the insulin granule with the cell membrane, most of the insulin is in crystalline form. The crystals are formed from hexamers of insulin complexed with 2 zinc atoms. [3 insulin dimers exist in a plane. Each dimer is held together by hydrogen bonds in a pleated sheet (AA24 and 26 in the B-chain.) The zinc atoms, one above and one below the plane, are coordinated with the B10 histidines ] . C peptide is in the soluble phase. References: 1) http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/pancrease /insulin_struct.htm 2) Katzung, Basic and Clinical Pharmacology, 8th ed, pp713 IIC. Comercial preparations of human insulin vary greatly the time until onset of action and the duration of action following injection. For regular insulin, NPH insulin, and ultralente insulin, what is different about their method of formulation that results in their different times of action? Answer: All these preparations of insulin are solutions or suspensions of heximeric insulin containing zinc in a buffer at neutral pH. The difference with NPH (Neutral Protamine Hagedorn) and regular insulin is that protamine is added to the crystals. The molecules contain about 1 molecule of protamine, one molecule of zinc and six insulin monomers. The point is that protamine stabilizes the crystal. The difference between ultralente and regular insulin is in the method of preparation and the excess zinc in the crystal. The principle is that insulin solubility is greatly reduced when more zinc molecules are present in the crystal than insulin monomers. A special method of crystal formation is required. References: 1) MDconsult search "NPH Insulin", "Ultralente Insulin" 2) Http://www.caerlas.demon.co.uk/insulin.htm 3) Cecil Textbook of Medicine, 20th ed, pp1264-66 4) Katzung, Basic and Clinical Pharmacology, 8th ed, pp717 IID. How do the insulin analogues Lispro, Aspart, and Glargine each differ in structure from humulin? What is the chemical and/or medical advantage of this insulin homolog for patients using an insulin pump? Answer: Lispro: Reversal of the sequence of residues at B28 and B29 decreases the tendency for beta-sheet interaction between insulin monomers. Inhibition of interaction inhibits fibrillation which is a major problem in the tubes of insulin pumps. Also, Lispro has a shorter onset of action and peak effect duration ( between 5-15 minutes: between 30-90 minutes) than regular insulin (between 30-45 minutes; between 2-4 hrs). This allows for injection just before eating instead of 30 to 45 minutes prior to eating. Aspart: is made by substituting aspartate for proline at B28. Its is similar to lispro. Glargine: Substituting one amino acid on the A-chain and adding two amino acids to the B-chain results in an analogue that is soluble at acidic pH but precipitates under the skin at neutral pH. This results in a very long acting insulin that can provide basal insulin for 24 hours. References: 1) www. postgradmed.com/issues/1997/02_97/bohannon.htm 2) www.prous.es/mom/jul_99/mom.html 3) Katzung, Basic and Clinical Pharmacology, 8th ed,

EXPLANATION FOR OBJECTIVE V: VA. Does insulin have a major effect upon glucose membrane transporters in the heart, striated muscle, brain, liver, adipose, lens, or red blood cell? Do glucose transporters facilitate active transport, secondary active transport, or passive diffusion? Answer: In general, insulin will increase the concentration of Glut-4 transporters in the cell membrane in heart, striated muscle, adipose tissue and most of the other cells of the body. Insulin does not increase the concentration of receptors found in brain, liver, lens, and red blood cells. In general, the entry of glucose from the blood into most cells is considered to be passive. That is, insulin can increase the rate of uptake, but the concentration of glucose in the cell can not exceed that of glucose in the blood. VB. Walley is learning that on weekends when he plays hard all morning, he has to adjust his insulin schedule. Explain the probable change and the biochemical basis f or the change. Answer: The insulin must be decreased or the result might be hypoglycemia. Exercise, a shortage of oxygen in muscle cells, or both causes more Glut-4 transporters to be inserted into the membrane. This increased insertion is in addition to the insertion as a result of insulin and independent of insulin. The result is increased glucose removal and a higher probability of hypoglycemia. VC. About 15 months after Walley started taking insulin, his mother noticed that he seemed to be more susceptible to hypoglycemia. Both Walley and his Mom are being very careful to adjust the dose for changes in diet and exercise. What happens to insulin dependent diabetics that helps to explain increased hypoglycemic episodes? Answer: When normal people experience hypoglycemia, with or without high blood insulin concentrations, the alpha cells release large amounts of glucagon. In insulin dependent diabetics, the alpha cells lose the ability to respond to hypoglycemia. this is thought to add to the chance that they will suffer hypoglycemic episodes. VD. One of the effects of having abnormally high blood glucose is demonstrated by the development of cataracts in diabetic patients. Find a theory that might explain the production of cataracts as a result of high blood glucose. Write the enzyme reaction(s) that help explain this theory. Answer: The most well worked out and accepted theory (not fact) is the production of high levels of sorbitol by the enzyme aldose reductase. The sorbitol, trapped in the cell, increases the osmotic pressure and causes swelling of the lens fiber cells. Eventually the cells rupture lose their transparency. The reaction is the first step in the polyol pathway. The polyol pathway follows: VE. . Explain why this pathway normally proceeds slowly at normal bold glucose concentrations and becomes dangerous at higher concentrations. (One sentence will do.) Would a one month old infant with decreased galactokinase activity due to genetic mutation be susceptible to cataract formation? Answer: The Km for aldose reductase is about 10 times the concentration of blood glucose. Answer: Yes! Large amounts of galactose are produced from lactose. The galactose would not be able to be phosphorylated and would reach high blood and cellular concentrations. Aldose reductase would produce galactitol in high concentrations. The same osmotic problems produced by sorbitol would result.

Type I Diabetes: Diabetes mellitus characterized by insulin deficiency, sudden onset, severe hyperglycemia, rapid progression to ketoacidosis, and death unless treated with insulin. The disease may occur at any age, but is most common in childhood or adolescence. All Insulin Solutions: are neutral solutions or neutral suspensions of zinc insulin containing 40 or 100 IU/mL. Hypoglycemia: An abnormally diminished concentration of glucose in the blood, which may lead to tremulousness, cold sweat, piloerection, hypothermia, and headache, accompanied by irritability, confusion, hallucinations, bizarre behavior, and ultimately, convulsions and coma. Diabetic Ketoacidosis: Complication of diabetes resulting from severe insulin deficiency coupled with an absolute or relative increase in glucagon concentration. The metabolic acidosis is caused by the breakdown of adipose stores and resulting increased levels of free fatty acids. Glucagon accelerates the oxidation of the free fatty acids producing excess ketone bodies (ketosis). Insulin Resistance: a state in which a given concentration of insulin produces a subnormal biological response. Most commonly refers to diminished effectiveness of insulin in lowering blood sugar levels but is just as important in all other types of metabolism. Arbitrarily defined as requiring 200 units or more of insulin per day to prevent hyperglycemia or ketosis. The molecuolar explanation for insulin resistance in type 2 diabetes remains unknown . Insulin-Like Growth Factor I : A well-characterized basic peptide believed to be secreted by the liver and to circulate in the blood. It has growth-regulating, insulin-like, and mitogenic activities. This growth factor has a major, but not absolute, dependence on SOMATOTROPIN. It is believed to be mainly active in adults in contrast to INSULIN-LIKE GROWTH FACTOR II, which is a major fetal growth factor. Insulin-Like Growth Factor II: A well-characterized neutral peptide believed to be secreted by the liver and to circulate in the blood. It has growth-regulating, insulin-like and mitogenic activities. The growth factor has a major, but not absolute, dependence on SOMATOTROPIN. It is believed to be a major fetal growth factor in contrast to INSULIN-LIKE GROWTH FACTOR I, which is a major growth factor in adults. Epitopes: Sites on the surface of an antigen molecule to which a single antibody molecule binds. An antigen has many different epitopes and reacts with antibodies of many different specificities. Diabetes Insipidus: A metabolic disorder due to disorders in the production or release of vasopressin. It is characterized by the chronic excretion of large amounts of low specific gravity urine and great thirst. Normal respiration rate 15 to 17/minute. What is the reaction of the sympathetic nervous to hypo or hyper glycemia? Hypoglycemia is covered in facilitated cases.

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