An Unexplained Death
By Justin Hines, Lafayette College and Marcy Osgood, University of New Mexico
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Instructor Notes for “An Unexplained Death”, Carbohydrate Metabolism Case Study
Topic Pre-requisites: Students should have exposure to the topics through Chapter 17 in Berg Biochemistry, 8th ed.
Overview
This case is designed to help students understand the importance of vitamins as precursors of coenzymes as well as to provide a context to review the reactions of glycolysis, the pentose phosphate pathway, the pyruvate dehydrogenase complex, and the citric acid cycle. As such, we have anticipated that students will have been exposed to material in the textbook through Chapter 17 of Berg Biochemistry, 8th ed. before beginning the case. Students may work individually or in groups to complete this case study. Students are constantly encouraged to refer to their textbook throughout the case, and internet access is permitted, although it is not necessary for the completion of the case. Students are required to iteratively acquire, analyze, and integrate data as they progress through the case and answer assessment questions found throughout the case. All assessment questions are automatically scored.
The case will also keep track of the number of investigations that students conduct and report this number to the instructor; students are made aware of this fact as a means to encourage careful consideration of investigation options and discourage guessing. It is entirely up to the instructor what to do with this information; we suggest potentially rewarding students who efficiently solve the case and/or penalizing students who obviously used blanket guessing (evident by the use of all or nearly all instigative options). For reference: there are 29 total investigative options within the case and 8 are minimally required to complete the case. We anticipate that careful students will typically make use of 12-20 options in solving this case.
Learning Objectives
This case is intended for remediating or extending these student capabilities:
1. The real-world application of the study of human metabolism. Student will:
2. Understanding of critical aspects of human central metabolism. Students will:
3. Practice critical thinking skills involving data. Students will:
Some questions are designed to show areas of difficulty for students
Suggested implementation
Below we describe two options for course implementation. The hybrid Online/In-class approach is recommended. Time required for students to complete the online case will vary by group depending on the level of discussion between each investigation. The case study can be started and stopped, and so it is recommended to give students a window of 2-3 days in which to complete the assignment.
Hybrid: Online/In-class: (recommended approach; ~30 minutes of class-time expected)
1. Share the case study link with your students to work online outside of class, preferably in pairs or groups of three. Assign the case study to be due before your next class meeting. Students should be instructed to bring copies of notes and/or printed answers to assessment questions to the following class period.
2. Review the online answers before the following class for difficult areas for students (see expected areas of difficulty above).
3. Lead students in a discussion in pairs, groups, or as a class (depending upon class size and instructor preference) to address unresolved difficulties (~30 minutes in-class time).
4. We recommend using the supplied assessment questions on exams or as homework assignments to reinforce the difficult concepts covered. Please see the document “Exam Questions Case 2- Carbohydrate Metabolism.”
Online only approach: (minimal in-class time required)
1. Share the case study link with your students to work online, preferably in pairs or groups of three. Assign the case study to be due before your next class meeting.
2. Review the online answers for difficult areas for students (see expected areas of difficulty above).
3. Mention or remediate tough points during a portion of lecture.
4. We recommend using the supplied assessment questions on exams or as homework assignments to reinforce the difficult concepts covered. Please see the document “Exam Questions Case 2- Carbohydrate Metabolism.”
Suggestions for in-class discussions (these questions may also be used in summative assessments such as exams or scored quizzes):
You may be missing vital information needed to sufficiently explain this incident. You must complete all investigations before proceeding to the final assessment questions.
You may be missing vital information needed to sufficiently explain this incident. You must complete all investigations before proceeding to the final assessment questions.
This activity has already been completed, however feel free to review the information contained within.
I am finished gathering information for this investigation and feel I am able to fully explain the reason for this man’s death in terms of a biochemical issue. I can fully justify and completely explain my reasoning based on the evidence I have gathered.
A 65-year-old man of Scandinavian descent was rushed to the Emergency Room of your local hospital after he was discovered unconscious by a family member in his home. The woman who dialed “911” told the dispatcher that the man, her brother, was the local librarian of the past 10 years and had no spouse or children. She reported that they had spoken the day before, and he had acted strangely on the phone. She says that she came by the house to check on him. Upon arriving at the hospital, he went into cardiac arrest and could not be resuscitated. Upon seeing the man, one of the physicians gasped, recognizing him as the winner of the state lottery from a few years prior. She had attended high school with the man and knew that he had a difficult time dealing with his new-found wealth; he preferred to keep to himself. You are shadowing the local coroner who would like your assistance in determining what might have caused the man’s sudden health failure. Because a substantial inheritance will pass to the sister who reported discovering the man unconscious, the police have ruled the death ‘suspicious’ and have asked you to be unusually thorough in your investigations to determine the specific cause of death.
With the assistance of the coroner, you may conduct additional investigations to gather information about the case. The goal of this exercise is to correctly solve the case without conducting completely unnecessary investigations; hence, you are encouraged to carefully consider the information you receive with each investigation and avoid haphazardly guessing. You will be scored on this exercise based on your answers to assessment questions found throughout the case and so you are STRONGLY encouraged to use your textbook to complete this exercise; you may also use the internet as necessary.
Please note that there is a minimum set of investigations that must be conducted in order to have all the necessary information to fully understand the case. The number of investigations you select will be recorded and reported to your instructor, so randomly guessing could adversely affect your score. You should be both thorough and thoughtful in conducting your investigation. Hint: we recommend that you first thoroughly exhaust the use of broader initial investigation options, like interviewing someone, before proceeding to test specific hypotheses by performing more specific tests for particular enzyme activities, for example.
RECOMMENDED INITIAL INVESTIGATIONS
Evaluate overall physical appearance including the presence of insect bites or other injuries
Results: The subject does not appear to be particularly physically fit and shows a moderate to high amount of abdominal fat and low muscle tone; however, this is not uncommon for an American male of his age. Notably, the subject’s legs are swollen and spongy to the touch. Otherwise, the subject appears normal for a 65 year old male.
Interview patient’s co-workers to determine dietary habits
Results: Coworkers describe him as forgetful and perpetually late to work, both likely due to his suspected alcohol consumption habits; they report that he often smelled of alcohol and kept a flask of whisky in his desk. Nonetheless, he was rarely negligent in his work and otherwise appeared healthy, so everyone you speak to is shocked at his sudden death. As far as anyone knows, he ate a normal diet, though no one recalls seeing him eat much. He was never seen smoking and gave no indications that he used any illicit drugs. However, the night janitor noted that the man had some trouble walking the last time he had seen him working in the library. When the janitor attempted to help him to a cab, he appeared confused as if he was unclear where he was and where he was going.
Conduct autopsy to evaluate condition of major internal organs
Results: An unusual amount of fluid was found throughout the body, causing swelling of tissues and lower extremities; excess fluid can be an indicator of cardiac malfunction. Nothing unusual was found in the man’s stomach; in fact, there was very little organic matter found within his digestive tract, indicating that he had not consumed much solid food in the past 24 hours. No blood or intestinal parasites were found. Most major organs looked normal considering the age and overall health of the subject; however, considerable fat deposition in the liver and cirrhosis (scarring, indicative of liver damage) was observed. Notably, liver in this condition could be associated with undiagnosed diabetes. The coroner concludes, however, that liver failure (due to cirrhosis) was NOT the cause of death; cause of death is declared to be heart failure for unknown reasons.
Important information about the biochemistry of the heart: The heart must constantly absorb molecules from the blood to produce ATP and continue contracting. The heart can use the following molecules for energy:
The specific pathways used to metabolize ketone bodies and fatty acids will be discussed in Chapter 22 of the textbook and are not necessarily important to this case; however, it is important to note that in order for the heart to use ketone bodies or fatty acids for ATP production, these molecules must be converted into acetyl-CoA which enters the citric acid cycle. Likewise, most of the ATP produced from the oxidation of glucose requires the pyruvate from glycolysis to be converted to acetyl-CoA and also metabolized through the citric acid cycle.
Test hair for common toxins (heavy metals and narcotics)
Results: No heavy metals or narcotics were detected.
Investigate medical history including current medications
Results: The man has a medical record on file. You find that he was negligent in getting annual physicals, having last been seen by a doctor 9 years and 5 years earlier. During both visits, the doctor made a note that he recommended the man reduce his alcohol consumption, increase his exercise regimen, and begin taking a multivitamin. The doctor noted that the man is likely an alcoholic, based on his answers to interview questions. Alcoholics often have nutritional deficiencies due to poor diet, low total food intake (consuming most calories in the form of ethanol), and the inhibition of vitamin absorption by the digestive system. High alcohol intake can also cause ketone body production because large quantities of ethanol are metabolized to acetyl groups present as acetyl-CoA. It is notable that the man must not have taken his doctor’s advice the first time it was given…it is unknown if he did anything differently after the second visit. No other relevant preexisting conditions were found and no medications were listed.
Test the blood and various tissues for common poisons
Results: No common poisons were found in a toxicology test.
SECONDARY INVESTIGATIONS
Determine Blood Serum Concentrations
Common electrolytes: Ca2+, K+, Na+, Cl-, PO43-
Results: All values are in normal ranges. (normal ranges: [Ca2+] = 8.5-10.5 mg/dL; [K+] = 3.5-5.0 meq/L; [Na+] = 135-145 meq/L; [Cl-] = 100-106 meq/L; total phosphorus = 2.6–4.5 mg/dL)
Common lipids: free fatty acids (FFAs) and triacylglycerides (TAGs)
Results: 450 mg/dL FFAs (normal range: 190-420 mg/dL); 200 mg/dL TAGs (normal range: 40-150 mg/dL)
Galactose
Results: [Gal] = 1.5 mg/dL (normal range: 0-6.0 mg/dL)
Glucose
Results: [Glc] = 75 mg/dL (normal range: 70-110 mg/dL)
H3O+ ions: blood pH
Results: pH = 7.0 (normal range: 7.35–7.45)
The discovery that the man’s blood is acidic has opened up three new investigation options! A number of factors can cause metabolic acidosis including increased levels of CO2, lactic acid, or ketone bodies in the blood. You might consider investigating whether the concentrations of any of these molecules were elevated in this man.
Determine blood levels of ketone bodies: acetoacetate as a marker
Results: detectable levels of acetoacetate were found; however, they do not indicate diabetic ketoacidosis (normal range: undetectable)
Determine blood levels of lactate, pyruvate, and TCA cycle intermediates
Results: [lactate] = 5.5 meq/L (normal range: 0.5-2.2 meq/L); [pyruvate] = 0.32 meq/L (normal range: 0 – 0.11 meq/L). You also find elevated levels of alanine (most likely resulting from high pyruvate levels) and very high levels of α-ketoglutarate (αKG) and glutamate (most likely resulting from high αKG levels).
Determine blood levels of O2 and CO2
Results: pO2 = 88 mmHg (normal range: 75-100mmHg); pCO2 = 41 mmHg (normal range: 35-45 mmHg)
Insulin
Results: [Insulin] = 10 mU/mL (normal range: 0-29 mU/mL)
Phenylalanine
Results: [Phe] = 0.5 mg/dL (normal range: 0-2.0 mg/dL)
Determine Urine Concentrations
phenyl-pyruvate (a phenylketone)
Results: undetectable levels (normal range: undetectable)
Specific Enzyme Tests
Aldolase
Results: [Aldolase] = 3.0 U/mL (normal range: 0-7 U/mL)
Conduct skin biopsy and send fibroblasts to center to test for Electron Transport Chain enzyme activities
Results: ETC enzyme activities were normal.
Creatine kinase (CK)
Results: [CK] = 100 U/L (normal range: 40-150 U/L)
Glucose 6-phosphate dehydrogenase (G6PD)
Results: [G6PD] = 8 U/g Hb (normal range: 5-13 U/g Hb)
Lactate dehydrogenase (LDH)
Results: [LDH] = 270 U/L (normal range: 110-210 U/L)
Pyruvate dehydrogenase (PDH)
Results: PDH complex activity= 0.6 nmol/min*mg (normal range: 2-2.5 nmol/min*mg)
Culture fibroblasts and send to a lab to investigate which enzymes of the Pyruvate Dehydrogenase Complex are defective
Results: The results of the lab work are shown in the table below:
Enzyme Component | Residual activity (% of normal) in cultured skin fibroblasts |
---|---|
PDH Complex Total | 28% |
E1 (pyruvate dehydrogenase) | 26% |
E2 (dihydrolipoyl transacetylase) | 100% |
E3 (dihydrolipoyl dehydrogenase) | 100% |
Assay for the amount of riboflavin in various tissues
Results: The amount of riboflavin was at the low end of the normal range.
Assay for the amount of niacin in various tissues
Results: The amount of niacin was at the low end of the normal range.
Assay for the amount of thiamine in various tissues
Results: The amount of thiamine was nearly undetectable; this indicates a severe deficiency.
Assay for the amount of lipoic acid in various tissues
Results: The amount of lipoic acid was at the low end of the normal range.
Assay for the amount of pantothenic acid in various tissues
Results: The amount of pantothenic acid was at the low end of the normal range.
I have reviewed my options and I still need help. I would like to hire an outside consultant to review this case to provide guidance about what I might be missing.
The consultant reviews your notes and gives you this helpful advice: In this case, it is important to consider the details that would cause abnormalities in this man’s physiological (metabolic) state at the time of his death. You should completely examine the state of his body, his dietary habits, his medical history, and possible problems he might have had that are evident in his blood. For example, you may or may not have discovered that his blood is acidic… what are the possible causes for this? You should also make sure that you continue as in depth as possible in these lines of investigation as it is possible that there is a singular and specific reason for the failure of this man’s heart.
You have now completed the investigations and can go on to answer the following questions.
Congratulations on completing this Case Study! The following Case Summary gives a full explanation of the man's death.
Case Summary
The man died due to thiamine deficiency (“wet beriberi”) caused by his alcoholism. Thiamine deficiency resulted in minimal functionality of E1 of the PDH complex and αKG DH, so the mitochondria within the heart (where there is high energy demand) were unable to supply the needed energy to properly sustain a heartbeat. Energy sources of the heart (fatty acids, ketone bodies, and glucose) require functioning enzymes of the citric acid cycle such as αKG DH. Oxidation of glucose also requires a functional PDH complex. Although this deficiency would presumably affect all tissues, small mishaps in the heart prove fatal. The heart does not store triglycerides or glycogen, and so it has only about 3-seconds worth of chemical energy stored as ATP and creatine monophosphate. After those are used up, cardiac muscle cells will cease to contract. The death would have been preventable if the man had reduced his alcohol consumption and been taking a multivitamin, or changed his diet to include vitamin- and nutrient-containing food, rather than consuming mostly alcohol.