Murder or Medical Mishap?

1. Murder or Medical Mishap? Death on the Metabolic Ward by Nancy M. Boury Department of Animal Science Iowa State University, Ames, IA

2. Background You have been chosen for a very competitive paid under-graduate clinical internship position (uCLIP). You spent your first weeks becoming a certified nurse assistant (CNA). You will be paid to work as a CNA at St. Visintainer’s, assisting the nurses on the suspected metabolic disorder’s acute-care floor. As part of your internship, you will also be allowed to follow Dr. Saccharo as he sees clinic patients. The doctor is an expert in enzyme deficiency disorders relating to glycolysis. Because you hope to become a family physician, you’re excited about the opportunity to learn about these rare metabolic disorders. To prepare for your first day on the metabolic ward, Dr. Saccharo requests that you research normal sugar metabolism, particularly glycolysis.

3. Monday Morning at the Metabolic Disorders Clinic After working all weekend on Dr. Saccharo’s assignment, you feel ready for anything – including Monday morning. As you ride up the elevator with your mentor and fellow interns, Dr. Saccharo asks a few questions to determine how prepared you are for this morning’s clinic hours.

4. Please get into your groups of four and take out your clickers

5. CQ#1: Which of the following enzymes catalyzes a reaction that produces ATP during glycolysis? • Hexokinase(HK) • Phosphoglucoseisomerase (PGI) • Phosphofructokinase (PFK) • Triosephophateisomerase (TPI) • PyruvateKinase (PK)

6. CQ#2: Which of the following enzymes breaks one 6-carbon sugar into two 3-carbon sugars? • Hexokinase (HK) • Phosphoglucoisomerase (PGI) • Phosphofructokinase (PFK) • Aldolase • Enolase

7. Dr. Saccharo’sReview of Glycolysis Glucose is an important source of chemical energy, but this energy must be converted from a large denomination of energy (~680 kilocalories per mole of glucose) to more usable forms, such as ATP (~7.3 kilocalories per mole). Imagine standing in front of a vending machine with a $100 bill vs a $1 bill.

8. Dr. Saccharo’sReview of Glucose Homeostasis • Your body (particularly your brain) needs glucose as fuel for cell processes. When you eat, your blood glucose levels will temporarily rise, signaling the pancreas to release insulin. When you have gone without food for several hours, your pancreas will release glucagon, which triggers the liver to release glucose from glycogen stores. • If your blood sugars are too high, your blood vessels are damaged and the organs they supply with blood are damaged as well. Consistently high levels of blood sugars lead to kidney, heart, liver, and brain injury over time. • If your blood sugars are too low, your brain will starve for energy. As a result, you could pass out, enter a coma, and potentially die in a matter of hours.

9. Fasting- Glucose Levels Low Fed – Blood Glucose Levels High Pancreas Releases Glucagon Pancreas Releases Insulin Liver Glucagon Receptors Body Cells have Insulin Receptors Glucose Levels Normal Liver Releases Glucose into the Blood Body Cells Take Up Glucose from the Blood

10. Dr. Saccharo’sReview of Glucose Homeostasis • Diabetics either have cells that don’t respond to insulin properly (Type II diabetes, or late-onset) or don’t have functional insulin produced (Type I diabetes, or juvenile). • This sugar balance, however, isn’t the only thing we worry about here in the metabolic disorder’s ward. There are genetic disorders where a person is missing an enzyme required to break down the glucose (or other sugar) once it gets into its target cell.

11. Once glucose enters the cell, hexokinase will add a phosphate group to the glucose. This gives the glucose a negative charge and traps it within the cell. • This addition of a phosphate group is the beginning of glycolysis, which is also known as the Embden Myerhof pathway.

12. Please trade papers within your groups and check each other’s homework

13. Embden-Myerhof Pathway

14. Clicker Survey: How many mistakes were in the paper you graded? • 0-1 • 2-3 • 4-5 • More than 5 What was the most common mistake made within your group?

15. Jamie, one of your fellow interns, is bothered because he knows the clinic frequently treats people with enzyme deficiency disorders. He asks Dr. Saccharo: If glycolysis is needed to use glucose, how can a person live without all the enzymes needed for glycolysis?

16. One reaction – many different enzymes • Isoenzymes use the same reactants to produce the same products. • They may have: • Different genes • Different tissue expression • Different developmental timing of expression • They will likely have slightly different affinities for their substrates.

17. Phosphofructokinase as an example • Three different forms & three different genes: • PFK – L is expressed in the liver; the gene for this isoenzyme is found on chromosome 21 • PFK – M is expressed in the muscle; the gene is found on chromosome 12 • PFK – P is expressed in platelets; the gene is found on chromosome 10 • Looking at the homework – this is the same reaction • Adds a 2nd phosphate to fructose-6-phophate to form fructose-1,6-bisphosphate

18. CQ#3: If a person would be deficient in all 3 forms of PFK, this person would __. • Have lower than normal glycogen stores. • Have more mitochondrial activity than normal. • Be dead. • Produce more energy per glucose molecule because glycolysis would be unregulated.

19. Time for the clinic to open… Dr. Saccharo asks the interns, “Any questions before we get started seeing patients?” Please keep your corrected homework on your desk to use as a reference as we discuss the patients.

20. Levels (+ = normal, - = decreased levels, ++ = increased levels) Patient #1: Ann is a teenager and avid golfer who was referred to the clinic after being refused at the blood drive and tiring easily on the high school golf course during practice.

21. CQ#4: Assuming that Dr. Saccharo is correctly assuming that Ann has a defect in glycolysis, what is the most likely defect based on the blood metabolic profile? • Hexokinase • Phosphofructokinase • Triosephosphate Isomerase • Pyruvate kinase • Aldolase

22. Patient #2: Marie is a 32-year-old mother of three complaining of fatigue and muscle cramps with exercise. She had always blamed her intolerance to exercise on her sedentary lifestyle. However, she recently joined a gym and, after a week of aerobics classes, went to her physician who ordered a series of blood tests. The blood work came back with abnormal results, leading to her muscle biopsy and referral to the metabolic clinic.

23. Marie’s Blood Test Results Levels (+ = normal, - = decreased levels, ++ = increased levels)

24. CQ#5: The muscle biopsy showed an excess of glycogen in the muscle tissues. What is the most likely enzyme deficiency? • Pyruvatekinase • Hexokinase • Phosphofructokinase • TriosephosphateIsomerase • Aldolase

25. CQ#6: If you wanted to test red blood cells for their ability to complete glycolysis, what compound would you try to detect? • Fructose-6-phosphate • Aldolase • Pyruvate • Phosphofructokinase • Dihydroxyacetone phosphate

26. CQ#7: If you had some of Ann’s red blood cells in a test tube, what compound could you add to enable these cells to produce pyruvate? • Glucose • Fructose-1,6,bisphosphate • Insulin • Glucagon • Fructose-6-phosphate

27. Patient #3: Leo is a 25-year-old accountant. He is not anemic, has perfect teeth, and experienced two fainting spells recently. One happened after accidentally drinking a non-diet soda, the other after eating “naturally sweetened” fruit salad. His dentist referred him to Dr. Saccharo’s clinic. Dr. Saccharo suspected Leo may have hereditary fructose intolerance (HFI) and recommended a closely monitored fructose tolerance test. Leo was scheduled for the test and an observation bed at the local hospital the next day.

28. Hereditary Fructose Intolerance • HFI is a recessive disorder where individuals with HFI lack a functional copy of Aldolase B. • Individuals with HFI normally become symptomatic with weaning – as babies switch from breast-milk with lactose to formula with sucrose or sorbitol. The HFI may go undetected until adulthood, with adults self-regulating their diet to be low in sugars. • They are able to metabolize glucose, because they have Aldolase A and C. However, in the liver, it is Aldolase B that breaks down fructose-1-phosphate, sending dihydroxyacetone phosphate and glyceraldehyde-3 phosphate to glycolytic pathway. Since HFI patients lack Aldolase B, they are unable to break down fructose.

29. Glycogen Fructose metabolism – In the liver Glycogen phsphorylase Inside the cell Blood stream Fructose -1-Phosphate Inhibits Fructokinase Fructose Aldolase B Glucose-6-phosphate DHAP G3P Dihydroxyacetone phosphate (DHAP) Glyceraldehyde-3-phosphate (G3P)

30. CQ#8: Glycogen phosphorylase is an enzyme required to release the glucose stored as glycogen. A person with HFI who ingested fructose would have: • High levels of blood fructose. • High levels of blood fructose-1-phosphate. • High levels of blood glucose. • Low levels of blood fructose. • Low levels of blood glucose.

31. Regulation of glycolysis • Glycolysis is reversible. Most of the enzymes can work to break down glucose (gycolysis) or build more glucose (gluconeogenesis). • Products of a particular enzymatic reaction will generally inhibit that enzyme. • Several points of control exist. For example: • Hexokinase is inhibited by glucose-6-phosphate and activity is boosted by free phosphate. • Phosphfructokinase is inhibited by ATP and fructose-1,6,-bisphosphate • Pyruvate kinase is inhibited by ATP, but activated by fructose-1,6,bisphosphate and phosphoenolpyruvate.

32. Tuesday at the Hospital When you arrive at the nurse’s station to start your shift the next day, you discover that Leo was found dead in his room shortly after lunch. “What happened?!?” you almost shout at Leo’s nurse.

33. Leo’s Last Morning Leo’s nurse is occupied with the charge nurse and security guard. You overhear her saying, “Just an hour ago, he was fine. He told me the fructose tolerance test was exhausting. He planned to nap until lunch. When I went to check his vitals, I saw him lying in his bed, dead.” You see Dr. Saccharo behind the desk, where he is reviewing Leo’s test results.

34. Fructose Tolerance Test Blood Glucose Levels

35. CQ#9: What was the effect of giving Leo intravenous fructose? • A rise in blood glucose levels. • A decrease in blood glucose levels. • An increase in glycogen stores. • A rapid drop in blood pressure.

36. Explanation of Test Results • The lack of Aldolase B causes a build-up of fructose-1-phosphate. • This will trap phosphate in the liver. • Fructose-1-phosphate will inhibit glycogen phosphorylase. • Glycogen phosphorylase is required for the liver to break glycogen into glucose-6-phosphate.

37. CQ#10: If Leo had been given too much fructose, he would be at serious risk of coma or death. If caught in time, what treatment would you recommend to rescue him? • Insulin • Glucagon • Glucose • Fructose • Phosphofructokinase

38. So what happened to Leo AFTER the test?

39. A few drops of blood were found in the sink and a syringe was found in the biohazard trash instead of the Sharps disposal bin. Dr. Saccharo is curious about the blood and the syringe.

40. While the hospital awaits the results of the DNA analysis of the blood and the chemical analysis of the syringe, Dr. Saccharo reserves a small sample of the blood to test for metabolic activity.

41. Dr. Saccharo’s blood test results Levels (+ = normal, - = decreased or absent levels, ++ = increased levels)

42. CQ#11: If the syringe in the medical waste in Leo’s room contained ___, there would be cause for a full-scale investigation. • Insulin • Glucagon • Fructose • Aldolase • Glucose

43. CQ#12: Dr. Saccharo reports his lab results to the police, saying in his expert opinion that they should look for someone with ___. • Pyruvate kinase deficiency • Type I diabetes • Phosphofructokinase deficiency • Glycogen storage disease • Hexokinase deficiency

44. Did Dr. Saccharo’s tip solve the case? Later that week, you pick up the newspaper and read that police arrested an individual for Leo’s murder. It seems that the murderer had some questionable business practices that Leo noticed during tax season. The murderer was also a patient at the clinic. He saw Leo walking into the clinic and overheard the nurse scheduling Leo’s fructose test. He knew the test was dangerous and thought his actions would be considered an unfortunate complication or medical accident.