NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
CASE TEACHING NOTES
for
“A Diet to Die For: An Exploration of Oxidative Phosphorylation”
by
Terry Platt, Department of Biology, University of Rochester
Eric Ribbens, Department of Biological Sciences, Western Illinois University
INTRODUCTION / BACKGROUND
This case study is a “clicker case.” It combines the use
of student personal response systems (clickers) with case
teaching methods and formats. The case is presented in
class using a series of PowerPoint slides in parts, or stages.
After each stage, students are asked to respond to questions
(called “clicker questions”) posed by the instructor. In this
way, the students work their way through the material to
understand the problem presented in the case. Specifically
designed for use in large introductory science classes, the
method integrates lecture material, case scenario material,
student discussion, and (clicker) questions.
The overall objective of this case is to engage
students in a story problem about the classic “uncoupler”
2,4-dinitropheonol (DNP) that leads them to a deeper
understanding of oxidative phosphorylation. The pre-
class reading (included at the end of these teaching notes)
presents historical background information on DNP, a
weight-loss drug that was used in the 1930s, often with
fatal consequences, leading to the establishment of the
Food and Drug Administration. The in-class scenario,
presented in the form of a PowerPoint presentation
(~2.17MB), describes a college athlete who uses
dinitrophenol obtained on the internet to lose weight,
with a life-threatening reaction that requires emergency
room treatment. The scientific reasons for the dangers
he encountered are revealed as his twin sister asks the
attending physician questions. These focus on the
unusual properties of DNP to promote understanding
of the crucial role of a proton gradient in coupling ATP
synthesis to electron transport.
The case ends with the finding that controlled use of
a related mechanism helps keep babies and bears warm.
An optional epilogue provides historical background for
Dr. Peter Mitchell’s controversial Nobel Prize winning
“Chemiosmotic Hypothesis” (1978)—a classic example of
the power of the scientific method. Topics that interface
with this case include scientific method (hypothesis
Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens
testing), toxicology, thermoregulation, dieting and
weight loss, public health, internet ethical issues, and the
history of science.
Designed for use in an introductory biology course,
this case may be useful in other courses such as physiology,
bioenergetics, biochemistry, cell biology, nutrition,
public health, history of science/scientific method, and
ethics.
Objectives
• Instill a historical appreciation for DNP’s early use as
a weight loss drug and its role in the formation of the
Food and Drug Administration as a regulatory agency.
• Use current issues of dieting and weight loss via
internet drugs to engage student interest in the toxic
effects of DNP.
• Challenge students to think about how inhibition
and uncoupling effects of drugs can deepen their
understanding of oxidative phosphorylation in
eukaryotic systems.
• Encourage students’ curiosity as to the chemical reasons
for DNP toxicity in the context of the chemiosmotic
hypothesis.
• Stimulate interest in further study by showing how
biological control of oxidative phosphorylation can
generate warmth in human babies, for example.
• Promote an overall understanding of the relationship
between ATP synthesis via oxidative phosphorylation
and electron transport in eukaryotic cells.
Concepts Covered
• Electron transport: the transfer of electrons through a
series of complexes in the mitochondrial inner mem-
brane in order to reduce molecular oxygen to water.
• Oxidative phosphorylation: the production of ATP by
mitochondrial inner membrane enzymes as a result
of the transfer of electrons derived from catabolic
oxidation.
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• In concert with the passage of electrons along the
electron transport chain, protons are pumped
outwards, resulting in establishment of an
electrochemical gradient.
• ATP synthesis within mitochondria is normally
“coupled” to electron transport, in that inhibiting
either process blocks the other one.
• Energy for ATP synthesis is directly derived from an
electrochemical proton gradient.
• Situations that “uncouple” oxidative phosphorylation
do so by dissipating or eliminating the proton gradient,
so that electron transport continues unabated but
ATP synthesis stops.
• Biological “uncoupling” by the protein thermogenin
is used by organisms to keep warm.
Misconceptions
• Diet products are safe and have no side effects.
• Any product you obtain through the internet or public
media sources cannot be harmful.
• The electron transport chain is directly connected to
ATP synthesis.
• The connection between electron transport and ATP
synthesis is mysterious.
• The transfer of electrons is obligatory for ATP
synthesis.
• Oxidative phosphorylation “uncouplers” allow ATP
synthesis without electron transport.
CLASSROOM MANAGEMENT
This case was designed to be used with personal response
systems (“clickers”) in large classes of 100 or more
students in an introductory biology course. It is designed
for one 75-minute class period, or may be expanded
slightly and spread over two sequential 50-minute classes.
Some advance reading and thinking by the students is
required. Student-student discussion in class may be
beneficial for some of the clicker questions or other
concepts.
Teaching the Case
Before the class period in which the case is presented,
students read the chapter in their text corresponding to
oxidative phosphorylation (which must not be confused
with photophosphorylation). They should also read the
Case Handout (included at the end of these teaching
notes) and think about the questions posed in it. This
account is based on factual observations from that time
in history—both the dyes and the munitions ingredients
Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
are uncouplers of oxidative phosphorylation, though
this was not known at the time (Parascandola, 1974).
In-Class PowerPoint Presentation
Slide 1: Title slide.
Slides 2–6: Charles, a college freshman wrestler,
purchases DNP (2,4-dinitrophenol) on the Internet in
order to lose weight, but things take a frightening turn
when he displays alarming symptoms and has to be
rushed to the Emergency Room.
Slides 7–10: Charles survives. His twin sister Cheryl
asks the attending physician a series of questions that
covers the major concepts of the case. Students learn
about the medical and historical aspects of DNP
use. This includes reference to two deaths described
in the literature, one due to a mere doubling of the
recommended dose.
Slide 11: Poses the central question of “What is
oxidative phosphorylation?” which students should be
familiar with from their Case Handout/assignment, and
where it fits in with cellular respiration overall. This is
an opportunity to remind students that this differs from
ATP made during glycolysis, for example, which is called
“substrate-level” phosphorylation.
Slide 12: Clicker Question 1 (CQ#1) is a review
question, and will determine whether students have a
basic understanding of what this central process actually is.
Slides 13–16: Review of oxidative phosphorylation
and how it is measured in vitro, setting the stage for
a question about what happens when you block the
process. A reminder here about “OILRIG” as an
acronym—“oxidation is loss, reduction is gain” (of
electrons), and that the electrons are donated to the
Electron Transport Chain (ETC) by the soluble electron
carriers NADH and FADH2 (derived from the vitamins
niacin and riboflavin).
Slide 17: CQ#2 tests whether students understand
from their reading that blocking the ETC with cyanide
(an inhibitor of Complex IV, cytochrome oxidase) will
also prevent the synthesis of ATP.
Slides 18–19: These slides answer the question
(yes, ATP synthesis is also blocked) and pose a parallel
question about inhibition with oligomycin, which
inhibits ATP synthase.
Slides 20–21: CQ#3 explores whether students
understand the meaning of “coupling”– that not only
does inhibiting electron transport prevent the synthesis
of ATP, but inhibiting ATP synthesis knocks out the
transport of electrons.
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Note to Instructors: It may be useful to have each
student provide an individual clicker answer first;
then, after you look at the distribution of answers,
have students discuss the question with neighboring
students to provide “collective” answers, and see whether
understanding has improved. This technique may be
used on subsequent questions as well.
Slides 22–23: Show that blocking ATP synthase
also prevents the ETC from continuing functionally,
and sets the stage for a structural glimpse of the ATP
synthase as a physical molecular motor. It is driven by
the passage of protons through a channel as favored by
their concentration and charge gradient (higher and
more positive outside the mitochondria), which turns a
motor that can thereby synthesize ATP.
Slides 24–25: Show the motor, driven by “proton
flux”—their origin from those that were pumped
outwards by the ETC, thereby creating a substantial
electrochemical potential.
Slides 26–28: Link the proton excess outside to the
action of ETC and to the ability of the mitochondria
to make ATP, culminating in a visual schematic of Peter
Mitchell’s Chemiosmotic Hypothesis.
Slide 29: Introduces DNP, which the students have
read about in their pre-class assignment in the context
of weight loss and establishing the Food and Drug
Administration, and asks what effect it might have on
“coupling.”
Slide 30: CQ#4 invites students to predict what will
happen when DNP is added to the “blocked” situation
outlined in CQ#3 previously. The answer is surprising—
oxygen is now consumed at a high rate, although no
ATP is made. This is called “uncoupling,” and puts DNP
in the category of other chemical “uncouplers” (like the
coal-tar dyes mentioned in the Case Handout), though
fails to explain why it has this effect. An extra credit
exercise might be to look at the structures of the two
mentioned dyes, dinitronaphthol and dinitrocresol.
Slides 31–33: These provide a visual depiction of
uncoupling, and delve into the chemical properties of
DNP to determine how it might work.
Slide 34: CQ#5 poses a hypothetical situation to
see whether students can predict that DNP (but not
HCl) will allow the “leakage” of protons across a lipid
bilayer to equilibrate a pH difference. This is relevant to
the high pH observed inside the mitochondrial matrix,
compared to outside, and thus why protons would be
energetically favored to move from outside to inside if
Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
provided a mechanism to do so (either through ATP
synthase or via DNP transport).
Slides 35–38: Provide the analogy of keeping a bicycle
tire inflated when it has a slow leak (continued pumping
is necessary). The ETC tries to compensate by pumping
protons ever faster, which requires faster electron
transport. In turn, this consumes a lot of oxygen (the
ultimate repository for those electrons), while failing to
restore the gradient (because of the DNP-caused “leak”).
This eliminates the means by which protons could drive
ATP synthase and generates instead a significant amount
of heat. Another analogy is revving a car engine without
the clutch being engaged: you burn a lot of gasoline
(consuming oxygen) and make a lot of heat from the
engine, but don’t go anywhere. As an “uncoupler” of
oxidative phosphorylation, DNP is very toxic.
Slides 39–40: Some animals have evolved a biological
strategy for harnessing this process through the use of an
uncoupling protein called thermogenin. It is a controlled
biological “uncoupler,” which functions, for example, to
keep human infants warm; at present, how its activity is
cellularly regulated to prevent runaway uncoupling like
DNP is not understood.
Note to Instructors: The presentation could end
here for many courses. For those where additional
time is available, the remaining slides offer a historical
glimpse at the origins of Peter Mitchell’s Chemiosmotic
Hypothesis (Mitchell, 1961). It flew in the face of
conventional thinking at the time, yet application of the
principles of “strong inference” (Platt, 1964)—testing
the predictions of alternative hypotheses—ultimately
vindicated the model, for which Mitchell received the
Nobel Prize in 1978.
Slides 41–43: These slides pose the favored
Chemical Coupling model of the time as a contrast to
the Chemiosmotic Hypothesis, and set the stage for an
experimental test that can distinguish between them.
Few current textbooks address this controversy (Scott
Freeman’s Biological Science, 3rd ed., 2008 is an exception),
which raged strongly for well over a decade, yet it is an
excellent demonstration of the importance of adhering
to scientific method to achieve scientific understanding.
Slides 44–47: CQ#6 has a complex question as its
“stem,” but is essentially asking whether ATP synthesis
can be driven solely by a chemiosmotic gradient; note
that no oxidizable substrate is present. After the shift
to the new buffer, valinomycin-mediated export of K+
makes the outside positive charge high, and the pH shift
makes the outside proton concentration high (by 100-
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fold), relative to inside. Protons are thus energetically
favored to move inside, and since the only channel
available to them is through the ATP synthase, ATP is
indeed synthesized. This is depicted in the figures and
described in the subsequent slides.
Note: Though elegant, this is a somewhat complex
experiment, and it may be helpful for students to work
for a few minutes with one or more neighbors to try to
predict the result. Several variations on this experiment
are presented in different introductory biology text
books, but all lead to the same conclusion, namely, that
ATP synthesis can be driven by a (sufficiently strong)
proton gradient, in the complete absence of any electron
transport.
Slide 48. Highlights Peter Mitchell’s adherence
to the principles of strong inference and alternative
hypothesis testing and the impact of his contributions
to the scientific achievements in the 20th century.
Slide 49: The gracious quote from Mitchell’s Nobel
address seems a fitting way to end the presentation.
POSTSCRIPT
One challenge to the overall chemiosmotic hypothesis is
the question of how the ETC manages to pump enough
protons out of the mitochondrial matrix into the much
larger volume of the cytosol, to maintain a sufficient
chemiosmotic gradient. Porcelli et al. (2005) provide
an interesting possible answer: their pH measurements
of the intermembrane space (pH 6.9) indicate that it is
considerably lower than the cytosol (pH 7.6), and that
the matrix itself is only about pH 7.8. The suggestion is
that the cristae are “pinched” (or even “plugged”?) at their
outer “ends” enough to hinder free equilibration with
the cytosol. This would sequester the pumped protons
in a much smaller volume than if they were dispersed
throughout the entire cytosol, and would establish the
appropriate electrochemical potential for ATP synthesis
much more readily than otherwise.
ANSWER KEY
Answers to the questions posed in the case study are
provided in a separate answer key. The answer key also
includes the answers to the pre-case assessment questions.
Those answers are password-protected. To access the
answers for this case, go to the key. You will be prompted
for a username and password. If you have not yet
registered with us, you can see whether you are eligible for
an account by reviewing our password policy and then
apply online or write to answerkey@sciencecases.org.
Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
REFERENCES
Print
Cornely, K. 1999. Case 23: The Role of Uncoupling
Proteins in Obesity. In: Cases in Biochemistry. New
York: John Wiley & Sons, p.75–77.
McFee et al. 2004. Dying to be thin: A dinitrophenol
related fatality. Vet. Hum. Toxicol. 46: 251–254.
Mitchell, P. 1961. Coupling of phosphorylation to
electron and hydrogen transfer by a chemi-osmotic
type of mechanism. Nature 191: 144–148.
Nelson, D.L., & Cox, M.M. 2008. Lehninger Principles
of Biochemistry. W. H. Freeman & Company, New
York, 5th ed. Figures from Chapter 19.
Nicholls, D.G., Bernson, V.S. & Heaton, G.M. 1978.
The identification of the component in the inner
membrane of brown adipose tissue mitochondria
responsible for regulating energy dissipation.
Experientia Suppl. 32: 89–93. PMID 348493.
Parascandola, J. 1974. Dinitrophenol and bioenergetics:
An historical perspective. Molecular and Cellular
Biochemistry 5: 69–77.
Platt, J.R. 1964. Strong inference. Science 146: 347–353.
Porcelli, A.M., Chelli, A., Zanna, C., Pinton, P., Rizzuto,
R. & Rugolo, M. 2005. pH difference across the
outer mitochondrial membrane measured with a
green fluorescent protein mutant. Biochem. Biophys.
Res. Comm. 326: 799–804.
Tainter, M.L., & Cutting, W.C. 1933. Febrile,
respiratory and some other actions of dinitrophenol.
J. Pharmacol. Exper. Ther. 48: 410–429.
Tewari et al. 2009. Weight loss and 2,4-dinitrophenol
poisoning. Brit. J. Anaesthesia 102, 566–567.
Internet
http://www.brookscole.com/chemistry_d/templates/
student_resources/shared_resources/animations/
ox i d a t i ve / ox i d a t i ve p h o s p h o r y l a t i o n . h t m l .
A fairly good animation.
Cartoons of the Chemiosmotic Theory:
http://stevebambas.com/images/AP%20220%20
ox%20phosph.jpg (Pearson copyright)
http://www.biologyreference.com/images/biol_03_
img0327.jpg
http://en.wikipedia.org/wiki/Thermogenin.
Non-shivering thermogenesis, used in human
infants, hibernating mammals (bears) and many
rodents.
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 NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

Acknowledgements: One author (T.P.) is indebted to

his friend and colleague Dr. Eugene Barber for expert
knowledge and sage advice in constructing this case, and
to his co-author (E.R.) for enormous patience, guidance
and perspective. We are both grateful to our many
colleagues in the “clicker case” group, and the reviewers
of this case for cogent and thorough comments as well as
constructive criticism and encouragement. Support for
developing this case was provided by NSF grant #DUE-
0618570.

•
Copyright held by the National Center for Case
Study Teaching in Science, University at Buffalo, State
University of New York. Originally published May 21,
2012. Please see our usage guidelines, which outline our
policy concerning permissible reproduction of this work.

Case Teaching Notes for “A Diet to Die For” by Platt and Ribbens Page 5
 NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

CASE HANDOUT
for
“A Diet to Die For: An Exploration of Oxidative Phosphorylation”
by
Terry Platt, Department of Biology, University of Rochester
Eric Ribbens, Department of Biological Sciences, Western Illinois University

“By the end of the two and a half decades between 1940 and 1965, the field of oxidative phosphorylation
was littered with the smouldering conceptual remains of numerous exploded energy-rich chemical
intermediates; the remarkable uncoupling action of 2,4-dinitrophenate [dinitrophenol] … remained
obscure; and the process of hypothesis-building, needed to keep faith with the chemical-coupling notion,
reached such fantastic proportions as to be hardly intelligible to those outside the field.”
—Peter Mitchell (1978 Nobel address)
Prologue: Coal-tar dyes were used in the late 19th century to color food; one favorite in Europe was Martius yellow
(dinitronaphthol), used in pastries and macaroni where the color implied the foods were rich in eggs. A related dye
with similar properties was Victoria Yellow (dinitocresol). Some deaths occurred due to the use of these dyes, which
called attention to their potential danger in food products. This led French and German scientists to study toxicity
of these coal-tar dyes with dogs: in moderate doses, the symptoms included increased respiration and rise in body
temperature (to 44° C in one case) before death ensued. Because of their toxicity, these two dyes were banned in
Europe for use in coloring food by 1900, and within a few years were similarly banned in the United States, although
seven other coal-tar dyes could legally continue to be used in food coloration.
During and after World War I, munitions factories in France commonly made explosives from 40% 2,4-dinitrophenol
(DNP) and 60% picric acid (trinitrophenol) rather than the trinitro-toluene (TNT) manufactured in Britain and
the U.S. Overweight workers in these factories were observed to lose weight, felt fatigued, and sometimes displayed
excessive sweating and elevated body temperatures. Various studies between 1910 and 1920 confirmed in animal
experiments the previously observed toxicity of dinitrophenol, without being able to identify the mechanism of its
action. One interesting possibility, that increased heat production was due to “a stimulation of cellular oxidation,”
became favored—a remarkable insight, considering that ATP itself was not discovered until 1929!
In the U.S., Stanford scientists learned of the early French studies and repeated many of those experiments with
similar outcomes. This appeared to present a genuine method for “free” weight loss, and by 1933 physicians had
carried out clinical studies, establishing dosage levels that were able to hold the metabolic rate of obese patients at a
level 40% higher than normal with no adverse effects. This resulted in weight loss without dieting, which was great
news for patent medicine promoters, who seized upon this finding with glee. They began marketing “miracle cures” for
obesity, and some physicians started to prescribe DNP as a weight loss remedy. By 1935, more than 100,000 people
were estimated to have been “treated” with DNP. Unfortunately, deaths attributable to DNP had begun to be reported.
Concern spread after a 1933 Newsweek headline blared “Diet and Die with Excess Alpha Dinitrophenol,” which told
“of a physician who had been ‘literally cooked to death’ when he took an overdose of the drug in an attempt to lose
weight rapidly” (Parascandola, 1974).
The amount of dinitrophenol that causes harmful effects varies among individuals, because the amount necessary to
realize moderate weight loss (therapeutic dose) and the amount that produces death (lethal dose) are separated by less
than a factor of ten. Given the propensity of patients to believe that “if one pill is good, two pills must be twice as
good,” it is easy to see how DNP was able to claim so many lives in so short a time. Common signs and symptoms in-
clude increased basal metabolic rate (the rate that you use energy at rest), rapid breathing, increased sweating, a feeling

Case Handout for “A Diet to Die For” by Platt and Ribbens Page 1
 NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

of warmth (even in chilly or cold conditions), and weight loss. Your heart rate, breathing rate, and body temperature
rise. Cataracts, skin rashes, and decreases in white blood cell counts are also associated with DNP ingestion.
At that time, the government had no control over “cosmetic” medications if no medical claims were made on the
label—and in any case “obesity” was not considered a disease. To a large extent, the deaths caused by misuse of DNP
forced the adoption of a new stricter “Food, Drug and Cosmetic Act” by Congress in 1938. Shortly thereafter, DNP
was banned as a weight loss drug by a newly formed governmental agency: the U.S. Food and Drug Administration
(FDA). This was one of its first official actions, and in fact the abuse of this “drug” may have been instrumental
in Congressional chartering of the FDA, an institution that we now take for granted. The current mandate of the
FDA has become to monitor for safety and efficacy “all food except for meat and poultry; all prescription and non-
prescription drugs; all blood products, vaccines, and tissues for transplantation; all medical equipment and all devices
that emit radiation, including microwave ovens; all animal drugs and feed, and even all cosmetics.” Yet even today,
DNP is available over the Internet.
It took another two to three decades to confirm that 2,4-dinitrophenol acted as an uncoupling agent in mitochondria
both in vitro and in vivo, though the mechanism of its action remained obscure until the late 1960’s. Due to the
pioneering efforts of Dr. Peter Mitchell, supported by those of his scientific colleagues open-minded enough to
consider his “Chemiosmotic Hypothesis” as a serious alternative to the “Chemical Coupling Hypothesis” (previously
assumed by many to be correct), we now understand how DNP works. In the class period(s) ahead you will explore
the mechanism of DNP action to illuminate the currently accepted cellular model for coupling between the electron
transport chain and ATP synthesis in the process called “oxidative phosphorylation.”
Background reading for class: The text chapter covering oxidative phosphorylation.
Ask yourself: 1. What is meant by “coupling” of oxidative phosphorylation?
2. What is meant by “uncoupling” of oxidative phosphorylation?
3. Under what kinds of conditions will these effects occur?

Case Handout for “A Diet to Die For” by Platt and Ribbens Page 2