Question

READ THE CASE STUDY AND ANSWER THE FOLLOWING QUESTIONS

2nd CASE: An Unexplained Death

A 65-year-old man of Scandinavian descent was rushed to the Emergency Room of your local hospital after a family member discovered him unconscious 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 been acting strangely on the phone. She says that she came by the house to check on him. After 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.

RECOMMENDED INITIAL INVESTIGATIONS:
Conduct autopsy to evaluate condition of major internal organs

Results: An unusual amount of fluid was found throughout the body; 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, a 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:

Fatty acids
Glucose
Ketone bodies (used when normal metabolism is failing)

The specific pathways used to metabolize ketone bodies and fatty acids will be discussed in Chapter 27 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 are converted into

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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.

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.

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.

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. 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 OF:

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

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Results: pH = 7.0 (normal range: 7.35–7.45)

The discovery that the man’s blood is acidic has opened up four 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.

The new options are:

Determine blood levels of ketone bodies: acetoacetate as a marker Determine blood levels of lactate, pyruvate, and TCA cycle intermediates

Determine blood levels of O2 and CO2 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)

SPECIFIC ENZYME TESTS: Aldolase

Results: [Aldolase] = 3.0 U/mL (normal range: 0-7 U/mL) 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)
1. The PDH complex is a complex of how many distinct enzymes must function sequentially to convert pyruvate

into acetyl-CoA? (Hint: You may wish to review page 333 of Tymoczko 3E before attempting this question.)

A. One
B. Two
C. Three D. Four
E. Eight F. Twelve

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The discovery that the man’s PDH complex activity is lower than normal has opened up a new investigation option! The new option is:

Culture fibroblasts and send to a lab to investigate which enzymes of the Pyruvate Dehydrogenase Complex are defective

SPECIAL INVESTIGATIONS –

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:
Residual activity (% of normal) in

Enzyme component

PDH Complex Total

E1 (pyruvate dehydrogenase component) E2 (dihydrolipoyl transacetylase)
E3 (dihydrolipoyl dehydrogenase)

cultured skin fibroblasts

28%

26% 100% 100%

2. Which of the following molecules are coenzymes that are essential for function of the PDH complex? (Select ALL that apply!)

A. Biotin
B. TPP (thiamine pyrophosphate) C. FAD
D. Tetrahydrofolate (THF)
E. NAD+
F. Vitamin B12
G. Lipoic acid
H. GDP
I. CoA
J. O2

The discovery that only E1 of the PDH complex has lower than normal activity has opened up five new investigation options! Consider the information about E1 (pyruvate dehydrogenase component) on page 334 and table 15.3 on page 272 of Tymoczko 3E before proceeding. The new options opened are:

Assay for the amount of riboflavin in various tissues
Assay for the amount of niacin in various tissues
Assay for the amount of thiamine in various tissues
Assay for the amount of lipoic acid in various tissues
Assay for the amount of pantothenic acid in various tissues

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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.

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 O2 and CO2
Results: PO2 = 88 mmHg (normal range: 75-100mmHg); PCO2 = 41 mmHg (normal range: 35-45 mmHg)

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 a-ketoglutarate (aKG) and glutamate (most likely resulting from high aKG levels).

3. Why might a person have elevated levels of pyruvate AND lactate? (Select ALL that apply!) Hint: Review pages 293-294 of Tymoczko 3E as needed to answer this question.

Because his/her body is doing lactic acid fermentation.

Because he/she is in a fasted state and his/her liver is running gluconeogenesis to produce pyruvate.

Because there might be a problem with the pyruvate dehydrogenase complex resulting in a surplus of pyruvate in excess of what is needed for lactic acid fermentation.

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4. The high levels of a-ketoglutarate could be explained by the loss of function of which enzyme?

Succinate dehydrogenase

Pyruvate kinase

Hexokinase

a-ketoglutarate dehydrogenase

Succinyl-CoA synthetase

Succinate dehydrogenase

Note: The simultaneous inactivation of both the pyruvate dehydrogenase complex and a-ketoglutarate dehydrogenase may be a critical clue to solving this case. Review page 325, the second ‘Clinical Insight’, and page 347 of Tymoczko 3E to look for the connection(s) between these two catalyzed reactions before continuing further in the case! You may also want to refer back to page 333 for information about the pyruvate dehydrogenase complex.

What are the similarities between the PDH complex and a-ketoglutarate dehydrogenase? (Select ALL that apply!)

They require the same coenzymes

They are both are part of the citric acid cycle

They both catalyze decarboxylation reactions

They both are part of glycolysis

They both catalyze oxidation reactions

They both catalyze reactions that proceed through a similar mechanism requiring three

specific enzymatic activities catalyzed by distinct subunits

Read pages 272-273 of the Tymoczko 3E and look at Tables 15.3 and 15.4 before answering the following

three questions: Many coenzymes are derived from what type of molecules? (Select ALL that apply!)

Salts

Carbohydrates

Proteins

Vitamins

Nucleic acids

Minerals

Which of the following coenzymes are essential for a-ketoglutarate dehydrogenase function? (Select ALL that apply!)

Biotin

TPP (thiamine pyrophosphate)

FAD

Tetrahydrofolate (THF)

NAD+

Vitamin B12

Lipoic acid

GDP

CoA

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8. What would be the most likely cause of a simultaneous loss of activity from both the pyruvate dehydrogenase complex and a-ketoglutarate dehydrogenase in this case?

Two genetic mutations must have recently occurred in this person

This person likely has two in-borne errors of metabolism--that is, genetic polymorphisms in the genes encoding these enzymes that have been present since birth

Heavy metal toxicity, such as, sudden exposure to mercury

Deficient O2 transport, resulting in hypoxia and a loss of cellular respiration

A vitamin deficiency

Dehydration

Acute alcohol intoxication

A lack of insulin action as a consequence of diabetes

Hormonal down-regulation of the expression of both enzymes

ENDING THE CASE AND TRIGGERING THE ASSESSMENT QUESTIONS:

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.

Conduct autopsy to evaluate condition of major internal organs

Interview patient’s co-workers to determine dietary habits

Investigate medical history including current medications Blood levels: H3O+ ions: blood pH

Determine blood levels of lactate, pyruvate, and TCA cycle intermediates Specific Enzyme Test: Pyruvate dehydrogenase (PDH)

Culture fibroblasts and send to a lab to investigate which enzymes of the Pyruvate Dehydrogenase Complex are defective

Assay for the amount of thiamine in various tissues Final Assessment Questions for “An Unexplained Death”:

9. Blood tests found elevated levels of lactate and pyruvate. You may or may not have also discovered that enzyme tests found elevated levels of lactate dehydrogenase (LDH) as well. What is a possible explanation for the elevated levels of all three molecules?

A. Because this man’s PDH enzymes are functioning deficiently, these enzymes require more pyruvate to function efficiently. LDH catalyzes the conversion of lactate to pyruvate, thereby elevating pyruvate levels in an attempt to counteract PDH deficiency

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B. Because this man’s PDH enzymes are functioning deficiently, there is a metabolic buildup of pyruvate. More LDH is produced to deal with the excess pyruvate. LDH catalyzes the conversion of pyruvate to lactate, thereby elevating lactate levels

C. Because this man’s PDH enzymes are functioning deficiently, these enzymes require more oxygen. Their consumption of oxygen creates a less oxygen rich environment elsewhere. More LDH is produced to catalyze anaerobic lactic acid fermentation. LDH catalyzes the conversion of pyruvate to lactate, thereby elevating lactate levels

10. Patients such as the one described in this case also often have very low transketolase activity. What is the connection between transketolase and the PDH complex?

Both participate in reactions that consume acetyl-CoA

Both require thiamine pyrophosphate as a coenzyme

Both require Ca2+ as a coenzyme

Both require pyruvate as a coenzyme

Both catalyze reactions that hydrolyze ATP

11. You were not able to look at a-ketoglutarate dehydrogenase activity directly in this case, but if you were able

to assay for it, would you expect to find that this man had reduced a-ketoglutarate dehydrogenase activity?

A. Yes

Not reduced but the same level of activity

No reason to predict either way

I expect he would have increased a-ketoglutarate dehydrogenase activity

12. Based on what you know about this case, which of the three catalytic subunits of a-ketoglutarate dehydrogenase would you expect to be rate limiting in this individual?

E1 (a-ketoglutarate dehydrogenase, requires TPP)

E2 (dihydrolipoyl succinyltransferase, requires lipoic acid and CoA)

E3 (dihydrolipoyl dehydrogenase, requires FAD and NAD+)

All three subunit activities should be equally impaired

No reason to predict any particular subunit will be impaired

13. Heart tissue mainly relies on fatty acids and glucose from the blood to produce ATP. Note that fatty acids are catabolized to produce acetyl-CoA, which will enter the citric acid cycle in heart tissue. Which of the following biochemical pathways are utilized by cardiac muscle to oxidize organic carbon molecules in order to produce ATP? (Select ALL that apply!)

A. Glycolysis

PDH complex

Citric acid cycle

D. Gluconeogenesis

14. Which biochemical pathways would be directly hindered by a thiamine deficiency? (Select ALL that apply!) (Hint: which of these pathways include reactions catalyzed by enzymes that require thiamine as a cofactor?)

Citric acid cycle

Glycolysis

PDH complex

Gluconeogenesis

Glycogenolysis

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15.

16.

17.

What would you expect to happen to [pyruvate], [a-ketoglutarate], and [succinyl-CoA] in cells that are thiamine deficient?

18.

19.

[pyruvate] decreases, [a-ketoglutarate] decreases, [succinyl-CoA] increases [pyruvate] decreases, [a-ketoglutarate] increases, [succinyl-CoA] decreases [pyruvate] increases, [a-ketoglutarate] increases, [succinyl-CoA] decreases

A. B. C. D.

in order to collect his lottery winnings as an inheritance?

Yes definitely! The evidence clearly points to murder

Maybe. The evidence points to foul play

Most likely not, the evidence points to another cause of death

Based on all the evidence you have gathered in this case, and your answers to previous questions, what do you think caused this man’s death? (Select ALL the relevant factors that should be included in a complete explanation of this man’s death!)

Murder

Vitamin deficiency due to alcoholism

Death due to acute alcohol intoxication

Thiamine deficiency

Inborn errors in the genes encoding the enzymes of the PDH complex

Diabetes mellitus (Type I or II)

Heavy metal exposure

Heart failure due to reduced activity of the PDH complex and citric acid cycle

Heart failure due to severe metabolic acidosis from lactic acid and ketone body accumulation

Heart failure due to diabetic ketoacidosis

How did excessive alcohol consumption likely contribute to thiamine deficiency (beriberi) in this case? (Select ALL that apply!)

Alcohol interferes with vitamin and nutrient absorption in the intestine by damaging the intestinal lining, so this man could absorb very little thiamine from meals

Alcohol was the majority of this man’s caloric intake, so his diet was lacking in key nutrients and vitamins such as thiamine

Alcohol can inhibit the ability of thiamine to act as a cofactor after it is absorbed, so very little of the already sparse dietary thiamine consumed by this man could bind to the enzyme

Alcohol can act directly as an inhibitor to key metabolic enzymes, so this man needed proportionately more thiamine than the average person

This man has a genetic disorder characterized by a mutation in the gene for transketolase, which results in a lowered affinity for TTP

How did thiamine deficiency result in this man’s death?

Because thiamine is an important source of energy for the heart, and the heart relies on the metabolic products of the PDH complex and the citric acid cycle, ATP production was inefficient, and unable to keep pace with the constant energy demand of contraction

Because thiamine is an important coenzyme for key enzymes in the metabolic pathways used by the heart, namely those of the PDH complex and the citric acid cycle, ATP production in this man’s heart was inefficient and unable to keep pace with the constant energy demand of contraction

[pyruvate] increases, [a-ketoglutarate] decreases, [succinyl-CoA] increases
Given everything that you now know about this case, do you think that a family member murdered this man

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C. Because thiamine can act as an inhibitor of key enzymes in the heart, namely those of the PDH complex and the citric acid cycle, ATP production in this man’s heart was inefficient and unable to keep pace with the constant energy demand of contraction

FINAL 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 a-ketoglutarate dehydrogenase, so within the heart (where there is high energy demand) the mitochondria 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 a-ketoglutarate dehydrogenase. 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 had been taking a multivitamin, or changed his diet to include vitamin- and nutrient-containing food, rather than consuming mostly alcohol.

EXTENDING YOUR UNDERSTANDING BEYOND THE CASE:
Another effect of chronic alcoholism is called “fatty liver.” Review the information found on pages 517-519 in

Tymoczko 3E to help explain why! Review that information and then answer the following question. 20. Why does fat accumulate in the liver due to alcohol consumption? Select all that apply.

The accumulation of FADH2 activates fatty acid synthesis in the liver

The accumulation of NADH and inhibition of gluconeogenesis activate fatty acid synthesis in the liver

Excess glycogenolysis in the liver

The inhibition of gluconeogenesis activates fatty acid degradation in the liver

The accumulation of glucose in the liver leads to fatty acid synthesis

Explanation: NADH accumulates due to the metabolism of ethanol through alcohol dehydrogenase and aldehyde dehydrogenase reactions. High levels of NADH inhibit gluconeogenesis and fatty acid oxidation, which leads to lactate accumulation and hypoglycemia due to the reverse reactions occurring. Since fatty acid oxidation normally generates NADH for oxidative phosphorylation, the excess NADH actually stimulates fatty acid synthesis instead.

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Answer 1