Lance Armstrong was certainly in the news this year, with the ongoing--and seemingly endless--saga of the USADA investigation into the use of performance enhancing drugs (PED) in professional cycling. I've gotten several inquiries about erythropoeitin (EPO) and thought that I'd write a short piece about the agent, its effects on the heart and vascular system, its use as a PED, and a little about the detection of its use.
The Hormone
The first thing to understand is that erythropoetin is a naturally occurring glycoprotein hormone. The existence of this hormone was theorized as early as the 1950's and the hormone was given the name erythropoeitin, but its isolation proved to be very difficult. The human body produces more than a half ton of blood in a lifetime, but only enough EPO to make a tiny pill. Early efforts sought to isolate the hormone from patients who were thought to have increased levels of EPO--those with anemia of various sorts. It was the serendipitous observation that excess erythropoeitin was excreted in the urine that eventually led to the isolation of the hormone. In 1977, Eugene Goldwasser from the University of Chicago isolated 8 mg of erythropoeitin from the dried concentrate of 2,550 liters of urine colleted from a group of aplastic anemia patients in Japan.
We know today that EPO is produced primarily in the kidney, and less so in the liver, and acts to stimulate production of red blood cells (RBCs) in the bone marrow.
The Blood
It helps with this discussion to have an understanding about blood itself. We often take for granted this liquid that courses through the body's arteries and veins. There is a cellular component to blood--cells that we typically call red or white. The RBCs far outnumber the white blood cells (WBCs), and it is the RBCs that are important for today's discussion. The non-cellular component of blood is the plasma, made up predominantly of water, but containing a large variety of proteins, minerals, and other substances.
The body produces RBCs in a process known as erythropoeisis. In adults, new RBCs are produced primarily in the bone marrow of the sternum (breast bone), vertebral column, and pelvic bones in a process that takes about a week. Immature RBCs are called reticulocytes and comprise about 1% of the total population of RBCs. RBCs have a lifespan of about 4 months and are then destroyed in the spleen or liver. The typical adult has about 20-30 trillion or so RBCs.
One measure of the amount of RBC's in the blood is called the hematocrit. If we take a small tube of blood, centrifuge it for a period, we'd be left with cells (mostly RBC's because they outnumber the other cells, by far) in the bottom of the tube and plasma at the top of the tube. The ratio of cells to plasma, expressed as a percentage, is the hematocrit. At my hospital's laboratory, the normal range for the hematocrit is 36.2% - 46.3% for adult men and 32.9% - 41.2% for adult women.
The RBCs contain the iron-containing protein, hemoglobin, that allows the red blood cells to carry oxygen to the body's tissues. Nearly 99% of the blood's oxygen is bound to the hemoglobin molecules; only about 1% is actually dissolved in the blood's plasma. In medical school, we learned that the RBCs with hemoglobin are the "box cars" of the train that delivers oxygen to the body's tissues.
The amount of hemoglobin in the blood can be measured. At my hospital's laboratory, the normal range is 11.9 - 15.4 g/dL for adult men and 10.6 - 13.5 g/dL for adult women.
There is a feedback mechanism in which low blood oxygen levels stimulate production of Epo by the kidney, which in turn stimulates production of RBCs in the bone marrow.
The Drug
The protein structure of EPO was worked out in the early 1980's and the agent was commercially available by 1985. The U.S. Food and Drug Administration (FDA) approved epoeitin alpha in June, 1989. Today, the agent is a made by genetic engineering techniques and produced in bacteria. The first commercially available product was Epogen, manufactured by Amgen Pharmaceuticals, but there are now several other formulations that are available.
One common brand of epoeitin alpha is Procrit, manufactured by Janssen Pharmaceuticals. I'll summarize the information provided by Janssen about their drug in their product insert, but the information will be similar for all of the formulations of epoeitin alpha.
Procrit is used for the treatment of anema due to: chronic kidney disease; the use of Zidovudine in patients with HIV infection; and the use of chemotherapy agents in patients with cancer. It is also indicated for reduction of RBC transfusion in patients undergoing elective, noncardiac, nonvascular surgery. The drug is an injectable agent that can be administered intravenously or by subcutaneous (beneath the skin) injection. The appropriate dosage depends upon the reason for its use.
Procrit produces an increase in the reticulocyte count within 10 days and an increase in the hematocrit and hemoglobin over a 2- to 6-week period. The rate of increase in each of these measured outcomes depends upon the dosage administered.
The use of agents like Procrit must be monitored carefully because there are potentially serious side effects or unwanted consequences, including myocardial infarction ("heart attack"), stroke, venous thromboembolism, thrombosis (clotting) of vascular access, tumor progression or recurrence, and even death. Indeed, the over-use of EPO would be implicated as a possible link to the deaths of 18 Dutch and Belgian cyclists from 1987 to 1990.
EPO as a Performance Enhancing Drug
Dating back to at least the 1960's athletes were aware of the potentially performance-enhancing effects of blood doping. You may recall that the 1968 Summer Olympic Games were held in Mexico City, at an elevation of 7,350 feet. It was recognized at those Games that athletes from higher altitudes performed well in the endurance events, presumably because of chronic adaptations to altitude that included an increased red blood cell mass. There are many descriptions of athletes using autologous transfusion (of their own banked blood) to enhance athletic performance in the subsequent couple decades. It wasn't until after the 1984 Olympic Games in Los Angeles that blood doping was banned by the International Olympic Committee (IOC). But as I mentioned above, it was in the mid-1980's that EPO became available and this would become a new avenue for increasing athletic performance.
The physiologic aspects of blood doping are worth considering for a moment, even in a simplistic fashion. In the endurance sports, athletes are limited by the amount of oxygen that can be delivered and used by the body's skeletal muscles. We might say that the aerobic capacity is related to the cardiac output (the amount of blood pumped per minute), the hemoglobin mass (the amount of hemoglobin in that blood), and the rate of oxygen extraction in the muscles. In the trained athlete, the hemoglobin mass might be the most easily influenced variable--one that is increased by blood doping or by the use of EPO.
In a previous blog post, I talked about the common heart-related medications that are included in the World Anti-Doping Agency's Prohibited List. In addition to banning blood doping--the transfusion or administration of blood, blood products, or blood substitutes--the Prohibited List in section S2.1 specifically bans erythropoeisis-stimulating agents such as EPO. All similar agents, as well as genetic methods related to erythropoeisis, are banned as well.
Detection of EPO
When Epo was first available, there was no method for detecting this PED in athletes. As an indirect method, professional cycling first conducted pre-race tests of the hematocrit, banning male athletes with a hematocrit >50% and female athletes with a hematocrit >47%. Keep in mind that in a retrospective study of blood donors in Denmark, 3.9% of non-athletes and 10.4% of elite rowers were found to have a hematocrit >51%. So measurement of the hematocrit alone is not a realistic way to identify use of EPO as a PED.
In 2000, the French national anti-doping laboratory developed a urine test that could identify the difference between an athlete's naturally-occurring endogenous EPO and synthetic EPO taken as a PED. This test took advantage of the the fact that each EPO type is actually a family of substances, each with the same protein structure but differing glycosylation, producing molecules of differing electric charge which were separable by the technique of electrophoresis.
This test was first used at the 2002 Salt Lake City Olympic Games, where 3 athletes (who had won 8 medals) were disqualified because of the detection of synthetic EPO in the urine.
In 2009, WADA has begun the use of a "biological passport" program to further enhance its ability to identify athletes who have used blood doping or the use of EPO. With this program, longitudinal profiles (over time) are kept of an athlete's blood-related parameters: hematocrit, hemoglobin, additional RBC parameters, the reticulocyte count, and serum EPO level. At its simplest, the measurements of serum EPO and reticulocyte count INCREASE after administration of synthetic EPO; these same measurements DECREASE after RBC transfusion or stopping the use of synthetic EPO. Unexplained changes in the parameters over time, particularly when linked temporally to competitions, can then be the evidence of doping.
