Blood Doping: Cheating Or Leveling the Playing Field?

STUDENT CASE STUDY—WOLFSON

Blood Doping: Cheating or Leveling the Playing Field?

Adele J. Wolfson, Schow Professor in the Physical and Natural Sciences; Professor of Chemistry, Wellesley College, Wellesley, MA

STUDENT CASE STUDY

Learning Objectives

Describe overall structure of proteins, including the amino acids that are linked to make polypeptides (proteins). Describe how proteins can be modified by addition of sugars. Describe how individual protein subunits can be combined into larger units.
Interpret oxygen-binding curves for hemoglobin and myoglobin and discuss how these are related to oxygen delivery.
Describe the steps involved in hormone signaling, particularly as related to erythropoietin (EPO).
Evaluate clinical data on EPO treatment of anemia.
Explain the importance of statistical variability in a sample, and how it relates to sample size
Assess methods for determining levels of EPO in body fluids. Explain how to distinguish between signal and noise.
Summarize data graphically.
Evaluate conflicting interpretations of scientific data.
Consider a body of evidence to make a recommendation regarding use of performance-enhancing drugs.

Preparatory material

Read carefully through this case study. You may also wish to refer to the Glossary in Appendix A for definitions of terms used in this case.
Review basic concepts of chemical bonding from previous chemistry course(s). Your instructor will suggest texts if you need additional background.
Watch Alex Gibney’s documentary, “The Armstrong Lie.” Your instructor will advise about which portions of the 2-hour film are most relevant.
Read Malcolm Gladwell’s article in the 9/9/13 issue of The New Yorker, “Man and Superman.” Access at

Introduction:

In 2012, Lance Armstrong was stripped of his sevenTour de France titles and quickly fell from his status as a venerated athlete to that of a despised cheater because of his use of erythropoietin (EPO), a drug that raises a person’s red blood cell count. Contrast this with the case of EeroMantyranta, a Finnish cross country skier, who in the 1960’s was honored as an Olympic champion, winning a total of seven medals. Mantyranta’s complexion was notably ruddy because he had a naturally high number of red blood cells in his circulation.

What is the difference (physiologically and ethically) between being born with extra red blood cells and taking a drug that increases your count? This case examines the data on EPO: how is it used medically and how effective is it? howis EPO detected in the body, and why is it so difficult to tell if an athlete has taken the drug? Along the way you will be introduced to the properties of the molecule EPO itself and how it acts, as well as to hemoglobin, the oxygen-transport protein of blood, which makes up 95% of protein in red blood cells. At the end of the case you will return to the question of the legitimacy of using naturally-occurring substances to enhance performance.

Exploring the Question[1]

Proteins

The two important molecule “players” in this case are hemoglobin (Hb) and erythropoietin (EPO). Hb is the oxygen-carrier in blood; it accounts for most of the content of red blood cells. EPO is a hormone that signals bone marrow to make more red blood cells.

Both Hb and EPO are proteins, one of the major classes of macromolecules in biological systems. As the name “macromolecule” implies, these are very large molecules with molecular weights ranging from thousands to hundreds of thousands. The synthesis of such large molecules would be a real problem for the cell if they were put together as a chemist might do it in the lab, bit by bit. Instead, for the most part, the macromolecules are put together from subunits, or monomers. So, the macromolecules are polymers made from monomers, of which there are a limited number, and these monomers are strung together by the same reaction over and over again. Proteins are biological macromolecules that are made up of one or more polypeptide, each of which is a chain of amino acids.

The monomers for proteins are amino acids. All amino acids have the overall structure shown in Figure 1:

(

Figure. 1. Generalized structure of an amino acid

There are twenty amino acids occur naturally in proteins. The letter “R” in the general structure refers to one of the 20 chemical groups that confer particular physical or chemical properties (charge, interactions with water, etc.) to each.

In a protein, amino acids are connected by (strong) covalent bonds called peptide bonds (Figure 2):

Figure 2. Generalized structure of a peptide

Based on its unique amino acid sequence, each polypeptide folds into the most stable 3-dimensional shape, and it is this shape that determines the specific function (activity) of the protein. The three-dimensional structure is stabilized by interactions called “non-covalent” because they are weaker than covalent ones, but they are still very important in the aggregate.

Some proteins have more than one chain (subunit), and these come together in specific ways that are also stabilized by non-covalent interactions. Hb is an example of one such protein. It is composed of four subunits, two of each of two kinds. Proteins with multiple subunits can be regulated in complex ways. More details of this regulation are discussed below.

Additionally, some proteins have additional “decoration” added to them after they are synthesized. EPO is an example of such a protein. It has sugar molecules added onto some of its amino acids. Sugars account for approximately 40% of its weight. As we will see in later sections, the pattern of these sugars can tell us about the origins of the EPO.

Many proteins also have non-amino acid, or “prosthetic” groups. One of these is heme, discussed on page 5, below.

Hemoglobin:

Examine the binding curves below (Figure 3) for hemoglobin and the related protein myoglobin (Mb). The units for pressure are “torr,” sometimes expressed as “mm Hg.” Typical oxygen pressure in the lungs is ~100 torr and in body tissues is ~40 torr. Note that “Percent saturated with oxygen” can also be described as “percent of subunits that have oxygen bound.”

Myoglobin can be considered an oxygen-storage protein in muscle tissue. Recall that hemoglobin circulates in the blood.Answer the questions that follow:

Figure 3. Oxygen-binding curves for myoglobin and hemoglobin

Questions

What is plotted on the x-axis? On the y-axis?
How does the percent saturation change with increasing oxygen concentration (O2 pressure) for both Mb and Hb?
What does this graph tell you about how hemoglobin effectively delivers oxygen from lungs to tissue?
We define p50 as the pressure (concentration) of oxygen when Hb or Mb is half-saturated. Is the p50 for Mb greater than or less than that for Hb?
How is p50 useful for quantification of Hb or Mb binding of oxygen?
On the graph, draw a new curve for hemoglobin that is shifted to the right. How does the value of p50 for the new curve compare to the original p50? How does the oxygen-binding affinity of the hemoglobin represented in the new curve compare to that of the original? What would that mean for delivery to the tissue?
How would shifting the curve to the left affect p50? In what part of the body would it be useful for this to occur?

The shape of the oxygen-binding curve of Hb is typical of “cooperative” binding. That is, binding of one molecule of O2 to Hb makes it easier to bind the next molecule of O2. Cooperativity almost always requires a protein with multiple subunits. The interactions among subunits, along with binding of some small regulatory molecules, lead to the cooperative effects. This behavior allows Hb to be fully loaded with O2 in the lungs, but to unload O2 to Mb in the tissues. Mb, with only a single subunit, does not display cooperativity.

The actual binding site for oxygen in Hb (and Mb) is the iron ion in the center of a molecule called “heme”; Figures 4a and 4b display this structure:

Figures 4a and 4b. Structure of the heme prosthetic group: a) chemical structure of the isolated group; b) Short-hand version of the ring structure with coordinated amino acid side chains in hemoglobin, with oxygen bound

Because iron is essential for synthesis of Hb, the increase in levels of Hb and red blood cells is accompanied by a decrease in stored iron from the storage protein ferritin, particularly in bone marrow, liver, and spleen.

There is one heme for each of the subunits in Hb, i.e.fourtotal. When oxygen binds to the iron in heme, it causes major changes in the overall structure of the protein, so much so that crystals of Hb will crack if oxygen is diffused in! The changes in structure on binding are responsible for much of the behavior of Hb related to binding oxygen in the lung and releasing it to the organs as needed.

Erythropoietin:

EPO is produced in the kidney in response to low oxygen levels and acts on bone marrow cells to produce red blood cells (Figure 5).

Figure 5. Physiological role of EPO

EPO is an example of a protein that signals cells to “turn on” specific genes and produce more of a particular product, usually a protein. Figure 6 presents a general scheme for such signaling molecules. As the diagram shows, the hormone or growth factor does not enter the cell, but rather attaches to a specific protein embedded in the outer membrane, called a receptor. Binding of the hormone or growth factor generates signals inside the cell that result in activation of one or more genes in the nucleus of the cell. Each gene is a specific DNA region that encodes the information for one polypeptide. When a gene is activated, thousands of copies of this genetic information are synthesized (“transcribed”) and transported to the cytoplasm of the cell, where they provide instructions for the synthesis of the polypeptide encoded by the gene.

Figure6.Generalized scheme for binding of a signaling molecule to the outer membrane, triggering events in the cells via second messengers (“relay molecules”)

More details for EPO are shown below in Figure 7.

Figure 7. Specific signaling used by EPO

JAK = Janus kinase and STAT = Signal transducer and activator of transcription. Note that there are many STATs.

Questions (Use Figure 5 to help answer these questions about Figures 6 and 7):

Using the nomenclature from the Fig. 6, above, what in Fig. 7 might be considered the “signaling molecule?” the “relay molecules”?
What do you expect is the target cell?

3. Which gene(s) do you expect will be turned on in these target cells?

As described above, EPO is a glycoprotein. The figure below shows that sugars are attached to the protein. This occurs through bonds to specific amino acids making up the protein. . In the figure, the "ribbon" represents the protein portion, while the other shapes identified in the key are different types of sugars (carbohydrates).

Bin Wu,JiehaoChen,J. David Warren,GongChen,ZihaoHua,Samuel J. Danishefsky. “Building complex glycoproteins: Development of a cysteine-free native chemical ligation protocol,” Agnew. Chem. Int. Ed. 45: 4116-4125. Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 8. EPO as naturally occurring, with sugars attached

The sugars do not seem to affect the way that EPO attaches to bone marrow cells to signal production of Hb and red blood cells, but they do influence how long EPO can circulate in the blood stream.

InterpretingHb and EPO levels in groups and individuals:

- Naturally-occurring levels:

The amount of hemoglobin in a person’s body is reported in g/dL (grams per deciliter, or 100 mL, of blood). The normal ranges are:

Women: 12.3-15.3 (according to some sources, 12-16) (mean 13.8) g/dL

Men: 14.0-17.5 (according to some sources, 13.5-18) (mean 15.7) g/dL

( original reference:Vajpayeet al, 2011, also Dailey, 1998 and 2001)

Questions:

Graph the values of hemoglobin for men and women in whatever format you think will convey the information.
How would you describe in words the differences between men and women? The differences within each of those groups?
If you were given a value for hemoglobin in a given sample, would that allow you to determine whether that sample came from a male or female? Why or why not?

You may also see reports of the hematocrit, the volume taken up by red blood cells compared to the total blood volume.

Now let’s look at EPO and hematocrit distributions in people with diagnosed illnesses, compared to normal values. Consider the graph below (Figure 9) and answer the questions that follow:

Other

Figure 9.Plasma EPO levels (milliunits/mL) in patients with different types and degrees of anemia and other conditions.

What is on the x-axis? On the y-axis?
Note that EPO levels are plotted on a logarithmic scale. What does this mean? Why would a researcher choose to use this scale?
Give the range for normal levels of EPO and for normal hematocrit readings.
Give the range for levels of EPO and for hematocrit readings for the condition identified here as “uremia.”
Give the range for levels of EPO and for hematocrit readings for the condition identified here as “PCV.”
If you were given a value for hematocrit for a particular patient, could you predict EPO levels in that patient?

Figure 10 takes a closer look at just two groups: those with anemia, and normal blood donors.

Figure 10. Plasma EPO levels in normal blood donors and patients with anemia.

Triangles represent normal donors, squares those of various anemias.

The dashed line represents the limit of detection of the assay.

Questions:

Compare Figure10 with Figure 9, above. What are the differences in units? In range?
What relationship between plasma EPO and hematocrit emerges more clearly from this figure than from Figure 9?
What is meant by the “limit of detection”? If the researchers obtained a value of plasma EPO of 2 U/liter, how confident could you be that this number is larger than 1 U/liter? If the researchers told you that EPO was absent from the sample, could you say with confidence that there was no EPO present? Sometimes, distinguishing between “nothing” and “some particular value” is referred to as the difference between “signal and noise.” Is this a good term for the phenomenon? What is the “signal” and what is the “noise?”

- Therapeutic use:

EPO has been used for at least 30 years to increase Hb and red blood cell production in patients with anemia.

Consider these data (Table 1) from one of the earliest reports of use of EPO in anemia:

From N. Engl. J. Med.,Eschbach, Joseph W. , Joan C.Egrie, Michael E Downing, Jeffrey K Browne, and John W Adamson,“Correction of the anemia of end-stage renal disease with recombinant human erythropoietin”, 316, 75. Copyright © 1987, Massachusetts Medical Society.Reprinted with permission from Massachusetts Medical Society.

Questions:

What is meant by “mean ± S.D.”? What does the “±” value tell you about precision of measurements?
The means in Table 1 are estimates based on the hematocrits measured for a limited number of patients. How would increasing the number of patients affect your certainty about how good these estimates are?
What appears to be the relationship between dose of EPO and hematocrit?
Why was it important to establish a base line before administering the therapeutic doses?

The patients entered into this study met the following criteria, among others:

-They were in a particular age range.

-They had hematocrit below a certain value.

-They had not lost blood due to any other reason than their anemia.

-They had no other diseases that might mask the effects of the treatment.

Questions:

Think about what question the investigators actually wanted to answer. What would the ideal (best case) data set look like to answer that question?
Given that the ideal experiment can never be done, what data set could they get that would allow them to estimate the answer?
Is their set of patients closer to “ideal” or to “data they could get”?

- Use of EPO in sports:

EPO has been banned from the Olympics since 1990. In order to determine whether or not athletes were using EPO illegally, officials needed methods to measure or otherwise detect its presence.

One way to measure EPO is to look for it in urine or blood. Natural and artificial EPO could be distinguished in the early days of its production because the sugars attached to the protein (see fig. 8) were slightly different when produced in the lab (in non-human cells) compared to those attached in the body. Some of these sugars have charges (positive or negative) associated with them, so that the overall protein moves differently in an electric field depending on the nature and number of these sugars[2]. The amount of EPO protein, natural or artificial, can then be quantified. Figure 11 shows an example of what the samples look like when subjected to the method:

Figure 11.Patterns of natural and artificial EPO obtained from urine

a: natural human EPO; b: recombinant (artificial) EPO source 1; c: recombinant (artificial) EPO source 2; d: urine from control subject; e and f: urine from two patients treated with recombinant EPO; g and h: urine from two cyclists in the Tour de France

Blood Doping: Cheating Or Leveling the Playing Field?

Bunn, H. Franklin. Cold Spring HarbPerspect Med 2013;3:a011619, ©, Cold Spring Harbor Laboratory Press.