Section: Introduction Part 1Overall Progress: 20%

Chapter 1. The Runner's Experiment

Lehninger Principles of Biochemistry
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The Runner's Experiment

By Justin Hines, Lafayette College and Marcy Osgood, University of New Mexico

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Race day had come. “Finally,” thought Michael. He was a marathon veteran, but this race was different. He felt terrible. “Probably a cold” he had told his girlfriend, but nothing was going to stop him today. Today was the day he would finally prove his brother Dave wrong, and he had 26.2 miles to do it.

The two young men shook hands shortly before the race started. Dave was thin, like Michael, but not “gaunt”. Michael’s girlfriend Jan had actually used that word to describe Michael a few days before. His cheeks had receded recently. “Seriously, you should stop this… you look terrible!” Jan said. “It’s just pre-race training… and of course, the experiment,” he thought. “I’ll be fine!” he assured her with a wink before leaving their apartment.

"Today is the day we settle this!"

“Today is the day we settle this!” he now called to Dave as they took off down the race route, but Dave only smiled and accelerated to leave Michael behind. As Dave disappeared into the crowd, Michael called out, “It’s not about speed! It’s about endurance dummy!”, but Dave was too far ahead to hear.

For weeks they had been talking about their plan, the experiment, and how much money they were going to make. Michael smiled to himself and then put his head down to focus on the run. Dave was long gone but Michael was certain that he would see him again soon enough… that is, until he started to feel dizzy…

Juan had worked at several marathon medical tents before. It was always the same: people try to run the race without training properly, and they end up at the tents. Most are dehydrated and exhausted, others just “hit the wall,” when their bodies run out of glycogen, and some even have heart attacks, mainly due to poor training. This particular day wasn’t very hot, but it didn’t take much to overwhelm people during a marathon. Working the races was a nice excuse for an ER doctor to get out in the sun for a few hours on the weekend and a chance to help some people… for Juan that was as addicting as running.

PHOTO CREDIT:

Two hours in and Juan was bored. The chatter on the radio was the same as always: dehydrated runners at both tents, and one elderly person from the crowd had to be treated for heat exhaustion, though it was really just from standing too long… so far it was a slow day.

Suddenly the radio chatter picked up. The ambulance from the medical tent at the 10-mile mark was headed in to his location at the finish line with a young man who was non-responsive. The incoming call was interrupted by a second voice: the ambulance from the medical tent at the 20-mile mark ALSO had a non-responsive man. ‘What are the odds?’ Juan thought. The two ambulances arrived simultaneously. Runner ID tags identified both subjects immediately: Michael and Dave Gard, two brothers! Dave was unconscious, but otherwise looked OK. When Juan saw Michael, however, he was startled into action; he would not have guessed that the two men were brothers!

You are a biochemistry student and you are shadowing an ER doctor who has just admitted two young males. One man, Dave, regained consciousness before arrival, whereas the other, Michael, regained consciousness only after arriving at the hospital and is still delirious. Neither man was particularly dehydrated, having drunk water during the race. Both have been stabilized, but blood and urine samples from before they were treated are available for you to test. It is up to you to discover what might be the problem with the two brothers.

Consider that there are two primary questions to answer:

  1. What caused both brothers to lose consciousness during the race? Here are some potential biochemical hypotheses for you to consider:
  2. Ketoacidosis
  3. Lactic acidosis
  4. Ammonia toxicity
  5. Mercury poisoning
  6. Acute hyperglycemia due to type II diabetes
  7. Hypoglycemia
  8. Phenylketonuria
  9. Maple-syrup urine disease
  10. What is the biochemical explanation for the differences in the conditions of the two brothers?

You may now conduct additional investigations to explore the details of this case and to test hypotheses so that you can eventually answer both questions. Note: for this case, you are encouraged to explore ALL possible investigations to gather as much information as possible to explain the case before finishing the case by continuing to the final case assessments.

RECOMMENDED INITIAL INVESTIGATIONS

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Evaluate the overall physical appearance of the two brothers, including insect bites or other injuries.

Results: The men are identical in height. Dave has a lean, athletic build, but is not unusually thin for a long-distance runner. Michael, on the other hand, appears to be severely emaciated. You note sunken eyes and cheek bones and protruding ribs, indicating a lack of not only body fat but also muscle tone. No injuries or other abnormalities are apparent.

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Investigate past medical history, including current medications.

Results: Neither man smokes, drinks, or uses illegal drugs. They are not on any medications. Dave reports that Michael had not been feeling well prior to the race, but had thought that he was “just coming down with a cold or something”. Given that the men were both avid marathoners, no one apart from Michael’s girlfriend Jan had been concerned about Michael’s recent and rapid weight-loss.

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Investigate the relationship between the two brothers in greater detail.

Results: Dave explains that the two men are not just brothers, they are best friends and despite their grossly different appearances at the moment, they are identical twins! He says, “Before we started our experiment just a few weeks ago, most people couldn’t tell us apart!”

Dave mentioned something about an experiment; you could ask him more about this. The following is now a new investigation option:

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Ask Dave about “The Experiment”.

Results: Dave tells you that the two brothers had been planning to start a new dietary supplement company, Gard Nutraceuticals, selling purified fish oil, which they believe is a health panacea. They disagreed on the best fish oil to bring to market however, so the men had been conducting an experiment to settle this disagreement. Both had been taking fish-oil pills along with a multi-vitamin for the past three weeks while they tapered back their training runs dramatically. When you press Dave about what else he and Michael were eating, shockingly he says “nothing”. They had been eating enough fish oil to consume 3000 Calories per day, which is normal for marathon training (approximately 330 grams of fish oil per day). Each brother had been touting a different product: Michael was taking oil from wild-caught salmon, while Dave was taking oil from a flathead (striped) mullet. The bet was to determine whether fish oil was an adequate caloric-replacement supplement for athletes, and whose product was better. To make the decision unambiguous, the pair was going to use the marathon to decide the winner since the boys had nearly identical marathon times in previous races.

The details of this fish-oil experiment may merit further investigation. In particular, some fish contain high levels of mercury, which could be toxic if consumed in large quantities. You now have the following two new investigation options available to you:

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Test hair for common toxins (heavy metals and narcotics) and ask Dave about mercury contamination in the supplements.

Results: Dave immediately points out that they worked with a chemist to extensively purify the fish oils to remove any mercury contamination. An independent laboratory verified that there are only trace levels of mercury left in their formulations, and Dave brings up the documentation on his smart-phone, showing that the analysis is good. Mercury toxicity will be negligible regardless of how much oil is consumed. Also, no heavy metals or narcotics were detected in hair samples from either brother.

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Investigate the composition of the dietary supplements the subjects were eating.

Results: Complete hydrolysis followed by esterification of the triacylglycerols (TAGs) in the two fish oil samples allowed fatty acid composition to be analyzed by gas chromatography.

The oil consumed by Dave (from the striped mullet Mugil cephalus) contained:

  • 11% 16:0
  • 5% 16:1(Δ7)
  • 15% 18:1(Δ9)
  • 36% 20-, 22-, or 24-carbon omega-3 or omega-6
  • 25% 15-, 17-, 19-, and 21-carbon fatty acids
  • 6% 12- or 14-carbon fatty acids
  • 2% unidentified

The oil consumed by Michael (from Atlantic salmon Salmo salar) contained:

  • 16% 16:0
  • 6% 16:1(Δ7)
  • 20% 18:1(Δ9)
  • 45% 20-, 22-, or 24-carbon omega-3 or omega-6
  • 4% 15-, 17-, 19-, and 21-carbon fatty acids
  • 7% 12- or 14-carbon fatty acids
  • 2% unidentified
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1.

What is the most significant difference between these two fish oil samples in terms of their fatty acid compositions? Note: it may be helpful to briefly review fatty acid structure and nomenclature from Chapter 10 (pages 357–359) of Lehninger, 6th ed., before answering this question.

A.
B.
C.
D.
E.
F.

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SECONDARY INVESTIGATIONS

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Determine Blood Serum Concentrations

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common immunoglobins (IgG, IgA, IgM) and ammonium (NH4+) levels

Results for Dave: All values for immunoglobin concentrations are at the low end of the normal ranges, which is normal for someone finishing a marathon. (normal ranges: [IgG] = 560–1800 mg/dL; [IgM] = 45–250 mg/dL; [IgA] = 100–400 mg/dL) [NH4+] = 20 mmol/L (normal range: 12–48 mmol/L)

Results for Michael: Severely low levels of IgG, IgM, and IgA. [NH4+] = 67 mmol/L (normal range: 12–48 mmol/L)

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5.

Which of the following might cause the concentration of ammonium ions found in the blood to increase? (Mark "yes" for all that apply.)

a. an increase in the rate of fatty acid catabolism by β-oxidation TrueFalse
b. an increase in the rate of protein and amino acid catabolism TrueFalse
c. an increase in the rate of glucose oxidation through the pentose phosphate pathway TrueFalse
d. an increase in the rate of glucose oxidation through glycolysis and the citric acid cycle TrueFalse
e. a defect in the urea cycle TrueFalse
f. galactosemia TrueFalse
g. lactose intolerance TrueFalse
Incorrect. You should have selected two answer options as true. Review Sections 18.1 and 18.2 (pages 695–710) of Lehninger, 6th ed., and please try again.
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free fatty acids (FFAs) and triacylglycerols (TAGs)

Results for Dave: 500 mg/dL FFAs (normal range: 190–420 mg/dL); 190 mg/dL TAGs (normal range: 40–150 mg/dL)

Results for Michael: 660 mg/dL FFAs (normal range: 190–420 mg/dL); 230 mg/dL TAGs (normal range: 40–150 mg/dL)

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glucose and glycated hemoglobin (HbA1c as a marker)

Results for Dave: [Glc] = 39 mg/dL (normal range: 70–110 mg/dL) Note: This value indicates severe hypoglycemia. HbA1c = 4.4% (normal range: 4–6.5%)

Results for Michael: 31 mg/dL (normal range: 70–110 mg/dL) Note: This value indicates severe hypoglycemia. HbA1c = 3.2% (normal range: 4–6.5%)

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6.

When a typical person runs a marathon, they do not become severely hypoglycemic to the extent that either Michael or Dave did. Some people consume carbohydrates during the race in the form of foods, gels, or sports drinks that have added sugar. However, even when dietary carbohydrates are not consumed during the race, how does the body of a healthy individual maintain adequate levels of blood glucose during sustained aerobic exercise? (Mark "yes" for all that apply.) Hint: You may wish to review pages 939–944 of Lehninger, 6th ed., with particular attention to the figures and to Table 23-2 before attempting this question!

a. Even-numbered fatty acids are catabolized and the carbon is used for gluconeogenesis in the liver. TrueFalse
b. Muscles run gluconeogenesis and export glucose into blood. TrueFalse
c. Ketone bodies are produced by adipocytes and converted into glucose by the liver. TrueFalse
d. Muscles convert lactate back into glucose and export this glucose back out into the blood. TrueFalse
e. The exclusively ketogenic amino acids (leucine and lysine) are deaminated and the carbon skeletons used to synthesize glucose. TrueFalse
f. Glucogenic amino acids are deaminated and the carbon skeletons used to synthesize glucose. TrueFalse
g. Most blood glucose will come from the breakdown of muscle glycogen (glycogenolysis) and the export of this glucose from muscles to the blood. TrueFalse
h. Most blood glucose will come from the breakdown of brain glycogen (glycogenolysis) and the export of this glucose from brain to the blood. TrueFalse
i. Most blood glucose will come from the breakdown of liver glycogen (glycogenolysis) and the export of this glucose from the liver to the blood. TrueFalse
Incorrect. You should have selected two answer options as true.
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H3O+ ions: blood pH

Results for Dave: pH = 7.31 (normal range: 7.35–7.45)

Results for Michael: pH = 7.2 (normal range: 7.35–7.45) The physician you are shadowing tells you that a value of 7.31 indicates acidosis but that this value will not normally cause a loss of consciousness. A pH value of 7.2 indicates severe acidosis and could result in neurological problems.

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ketone bodies (acetoacetate and acetone)

Results for Dave: low but detectable levels (normal range: undetectable)

Results for Michael: dangerously high levels of both found

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7.

What macromolecules can be catabolized such that the resulting carbon can be used to create ketone bodies?

A.
B.
C.
D.
E.

Hint: There is a better answer than this one. Review ketone body formation on pages 686–688 of Lehninger, 6th ed., and please try again.
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lactate and pyruvate

Results for Dave: [lactate] = 2.0 meq/L (normal range: 0.5-2.2 meq/L); [pyruvate] = 0.05 meq/L (normal range: 0–0.11 meq/L)

Results for Michael: [lactate] = 0.7 meq/L (normal range: 0.5–2.2 meq/L); [pyruvate] = 0.02 meq/L (normal range: 0–0.11 meq/L);

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Determine Urine Concentrations

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branched-chain α -keto acids

Results for both Dave and Michael: undetectable levels (normal range: undetectable)

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9.

What would an increased amount of branched-chain a-keto acids in the urine indicate? (Mark "yes" for all that apply.)

a. a defect in fatty acid catabolism TrueFalse
b. a defect in fatty acid transport TrueFalse
c. a defect in a citric acid cycle enzyme TrueFalse
d. a defect in carbohydrate metabolism TrueFalse
e. a defect in amino acid catabolism TrueFalse
f. phenylketonuria TrueFalse
g. lactose intolerance TrueFalse
h. maple-syrup urine disease TrueFalse
Incorrect. You should have selected two answer options as true. Review Section 18.3 (pages 710–725) of Lehninger, 6th ed., and please try again.
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phenylpyruvate (a phenylketone)

Results for both Dave and Michael: undetectable levels (normal range: undetectable)

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10.

What would an increased amount of phenylpyruvate in the urine indicate? (Mark "yes" for all that apply.)

a. a defect in fatty acid catabolism TrueFalse
b. a defect in fatty acid transport TrueFalse
c. a defect in a citric acid cycle enzyme TrueFalse
d. a defect in carbohydrate metabolism TrueFalse
e. a defect in amino acid catabolism TrueFalse
f. phenylketonuria TrueFalse
g. lactose intolerance TrueFalse
h. maple-syrup urine disease TrueFalse
Incorrect. You should have selected two answer options as true. Review Section 18.3 (pages 710–725) of Lehninger, 6th ed., and please try again.
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Specific Enzyme Tests

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lactate dehydrogenase (LDH)

Results for both Dave and Michael: [LDH] = 150 U/L (normal range: 110–210 U/L)

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liver Asp aminotransferase (AST) and Ala aminotransferase (ALT)

Results for Dave: Both enzymes are within the normal range (normal range: 7–55 U/L)

Results for Michael: Both enzyme levels are elevated.

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pyruvate dehydrogenase (PDH)

Results for both Dave and Michael: PDH complex activity= 2.5 nmol/min*mg (normal range: 2–2.5 nmol/min*mg)

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test cells for electron transport chain enzyme activities

Results for both Dave and Michael: ETC enzyme activities were normal.

You have now completed the investigations and can go on to answer the following questions, which will give you the opportunity to demonstrate your understanding of Dave’s and Michael’s conditions.

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11.

The citric acid cycle begins with the condensation of acetyl-CoA with oxaloacetate. Possible sources for carbon that may be converted into acetyl-CoA in active muscle include: (Mark "yes" for ALL that apply!)

a. pyruvate TrueFalse
b. β-oxidation of fatty acids TrueFalse
c. amino acid catabolism TrueFalse
d. conversion of ketone bodies from the blood back to acetyl-CoA TrueFalse
e. stored glycogen in the muscle TrueFalse
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Incorrect. You should have selected five answer options as true. Please review pages 686–687 of Lehninger, 6th ed., for information about ketone bodies and Table 23-2 (page 942 of Lehninger, 6th ed,) to review other possible sources of acetyl-CoA and try again.
Correct.
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Congratulations on completing this Case Study! The following Case Summary gives a full explanations of the two brothers’ conditions.

Case Summary

Because the human body cannot create glucose from the oxidation of even-chain fatty acids, both runners are starving their bodies of glucose due to their restrictive diets. Most of their energy is coming from fatty acid oxidation, so ketone bodies are being produced to free up CoA for more β-oxidation. Because their diet lacks carbohydrates and glucogenic amino acids, their glycogen levels will be very low before the race, regardless of their caloric intake. Their blood sugar is low and their bodies are compensating through protein wasting, resulting in their thin and emaciated appearances, and through ketone body production, resulting in acidified blood.

The differences between the two brothers’ conditions are not due to genetic polymorphisms (they are identical twins), but rather, arise from the composition of odd- vs. even-numbered fatty acids in their respective supplements. Even-chain fatty acids are NOT gluconeogenic precursors because the product of even-numbered fatty acid chain oxidation is multiple 2-carbon acetyl groups bound to CoA. Humans lack the enzymes necessary to use acetyl-CoA as a gluconeogenic precursor. In contrast, odd-numbered fatty acid catabolism produces one succinate (a 4-carbon citric acid cycle intermediate) for each fatty acid molecule oxidized. The result is that two glucose molecules (12 carbons total) can be produced from the oxidation of every three odd-numbered fatty acids.

Dave’s case is milder because the supplement he is taking contains a significant amount of odd-numbered fatty acids, and some of these may be used to produce glucose. Michael’s supplement has almost no odd-numbered fatty acids, so his body is undergoing more protein wasting (resulting in lower levels of non-essential body proteins like immunoglobins), and producing dangerous levels of ketones.

Ketone bodies are being formed BOTH from the products of β-oxidation of the fatty acids from the ingested fish oil and from ketogenic amino acids released from protein catabolism. Michael’s levels are higher than Dave’s because his body is undergoing protein wasting at a greater rate. Higher rates of protein wasting are the reason for Michael’s elevated levels of Asp aminotransferase (AST) and Ala aminotransferase (ALT). These enzymes (and additional aminotransfereases) are being produced in greater quantities to carry out the massive amount of amino acid deamination that must occur under these conditions. As a result of excessive protein degradation, Michael is also experiencing mild ammonia toxicity (in addition to all his other problems!). Ammonia passes readily through the blood-brain barrier and is highly toxic to the brain.