By David N. Leff
Just a few days from now, the International Cycling Union will rule on the validity of a blood-boosting urine test that fingers athletes who'll do anything to win.
For over a decade, racing cyclists, marathon runners and other competitors who strive for speed, endurance and bursts of energy have shot up the recombinant hormone erythropoietin (EPO). In its natural form, this glycoprotein, made in the kidney, signals the bone marrow to step up production of new, hemoglobin-filled red blood cells (RBCs) whenever levels of oxygen in body tissues drop too low.
On June 1, 1989, the FDA approved a recombinant version of EPO, synthesized by Amgen Inc. of Thousand Oaks, Calif. Trade-named Epogen, this injectable drug has become a multi-billion-dollar product. It originally was developed to counteract the chronic anemia in patients undergoing kidney dialysis, caused by their insufficient EPO production. This depleted red blood cells, and thus starved them of energy-supplying oxygen.
Recombinant rEPO went on sale in 30 European countries several years before its U.S. launch, and at once attracted the notice of athletes and their team physicians. They had been illicitly using anabolic steroids to increase muscle mass and enhance strength and endurance. There was one catch: That disqualifying fix could be detected readily in urine.
EPO escaped this give-away because its recombinant and natural molecular forms were identical - undistinguishable. So rEPO quickly became the blood-booster of choice in competition sports. But it had a deadly downside.
In February 1990, a 27-year-old Dutch cyclist named Johannes Dreaijer died after a race - allegedly of heart failure. More likely, according to medical experts, it was kidney failure due to rEPO. Since then, an unreported number of similar rEPO deaths are presumed to have occurred, without damping the urge of aficionados to win at all costs.
Tour de France Comes Cropper Over rEPO
It was this pervasive, undetectable abuse of rEPO that brought the 1998 Tour de France international bike race to its ignominious crash. Entire national teams were pulled from the event when packages and syringes of the recombinant hormone turned up in hotel rooms, automobiles and pockets. Many participants confessed to the French police or the Tour's organizers. Fewer than 100 riders of the original 189 finished the race.
In the Paris suburb of Chatenay-Malabry, the French National Anti-Doping Control Laboratory, which is accredited by the International Olympic Committee, was charged with devising a foolproof method of distinguishing rEPO from the natural hormone in time for the Sidney, Australia, Olympic Games in September 2000. Today's issue of Nature, dated June 8, 2000, carries the lab's initial report under the title "Recombinant erythropoietin in urine: An artificial hormone taken to boost athletic performance can now be detected." The paper's lead author is medical doctor and biologist Frangoise Lasne, who heads the laboratory's biology department.
The technology she and her co-author adapted to solve the molecular puzzle is a well-known technique of physical chemistry called isoelectric focusing. It separates mixtures of protein molecules into their components by subjecting them to an electric field having a previously established pH gradient.
Telling Natural EPO Sheep From rEPO Goats
"Isoelectric focusing," Lasne told BioWorld Today, "is a sort of electrophoresis, but whereas electrophoresis uses a determined pH, isoelectric focusing is performed in a pH gradient. The two forms are very similar," she explained, "but there are differences in their sugars, because these glycoprotein molecules are composed of a protein part and a sugar part. There are some differences in the sugar component, and these isoforms induce differences in their electrical charge. Isoelectric focusing enables us to show this difference of electrical charge.
"This is not a discovery," Lasne pointed out. "The originality - the difficulty - was to elaborate a procedure enabling us to compare the isoforms of the two hormones. What's new is our analytical procedure to see these differences."
Illicit Blood Doping? Seeing Is Believing
Lasne listed the three main steps in the test procedure, followed by a visible read-out of the result:
¿ "First, we must concentrate the urine sample, because EPO is at a very low level in urine;
¿ "The second step is isolectric focusing to separate the excreted EPO isoforms;
¿ "Third, a specific revelation of EPO [is required], because EPO is not the only protein in urine. There are lots and lots of other proteins. And our specific procedure reveals only the EPO.
¿ "The final result is to visualize the isoforms, as graphically illustrated in our Nature paper. They compose an isoelectric pattern of recombinant EPO, which differs from that of the natural hormone."
In the wake of the scandal-ridden 1998 Tour de France, the Anti-Doping Laboratory took 102 urine samples collected during the event out of deep freeze and subjected them to conventional EPO assay. "We found," Lasne recounted, "that 28 of these 102 samples presented very high levels of EPO. We chose the 14 with the highest levels for isoelectric focusing, because they were the most suspect of containing exogenous - that is, recombinant - erythropoietin. And indeed all 14 were positive - showing a banding pattern typical of the recombinant hormone."
"We also performed the EPO determination on about 100 urine samples during the 1999 Tour de France," Lasne went on. "Their levels were lower than in 1998. I thought it was because EPO doping was less prevalent. We have not performed our test on these samples, just determined their EPO levels."
The Anti-Doping Laboratory, instead, is engaged in a large-scale, blinded, placebo-controlled trial of some 1,000 subjects, in collaboration with facilities in Australia, Canada and Norway.
"When the International Cycling Union makes a decision about our test near the 20th of June," Lasne observed, "it will decide to use the test if three conditions are met:
"First, publication in a scientific journal - for which we have the Nature article.
"Second, to give them our results, using 200 samples from our large-scale validation study.
"And third, they will base their decision on the recommendations of an ad hoc scientific review panel, named by the International Olympic Committee to analyze our results."