Many people like to talk about drug use in sports, but I rarely see discussions about the science of anti-doping. In this article, I want to explain the science behind how testosterone is detected in urine and how athletes get away with taking testosterone.
The current test for testosterone is the testosterone to epitestosterone ratio test (T/E ratio) alongside the carbon isotope ratio test.
To understand how the T/E ratio test works you first have to understand how testosterone is produced in the body. Cholesterol is the starting substance for the formation of testosterone. As you can see in the diagram, there are several enzymes responsible for the conversion of cholesterol to testosterone. These starting substances (precursors) are transformed into testosterone.
A byproduct of cholesterol metabolism is formed called epitestosterone. This is very similar in structure to testosterone, but it is inactive. It only differs in the orientation of the hydroxyl group:
Testosterone and epitestosterone cannot be excreted in the urine because they’re hydrophobic, meaning they are not soluble in urine (like how oil separates in water). So during metabolism enzymes add glucuronic acid to testosterone and epitestosterone (glucuronidation) making them more water-soluble allowing them to be excreted in the urine.
Normally testosterone and epitestosterone are excreted in a 1:1 ratio in the urine, so when someone takes testosterone the ratio increases (because the amount of epitestosterone excreted will not change), if the ratio exceeds 4:1 then the athlete is suspected of doping.
However, this test depends on genetics. Some people naturally have a ratio above 4:1. To confirm if the person was taking testosterone, anti-doping labs perform a carbon isotope ratio test.
Carbon-12 is the most common isotope of carbon-containing 6 neutrons and 6 protons. Carbon-13 differs only by having an additional neutron. The basis of this test is that synthetic testosterone has less carbon-13 than testosterone produced in the body. So anti-doping labs determine the 13C/12C ratio value of testosterone in the urine to see whether it’s natural (endogenous) or synthetic (exogenous) testosterone.
So this means a low carbon-13 to carbon-12 ratio confirms someone is using testosterone.
Now there are many flaws of the T/E ratio test. For one, the natural ratio of testosterone to epitestosterone can vary because of genetics. Some people can have a ratio that is far lower, this is because they lack an enzyme which excretes testosterone in the urine. A study found differences in urinary testosterone excretion in Korean and Swedish men.
So why do some people lack this enzyme to excrete testosterone in the urine? Everyone has two copies of each gene, one from each parent. You can have both copies of a particular gene (insertion/insertion polymorphism), lack a single copy of the gene (deletion/insertion polymorphism), or you can lack two copies of the gene (deletion/deletion polymorphism).
The UGT2B17 enzyme (UDP-glucuronosyltransferase 2B17) adds glucuronic acid to testosterone. If a person lacks two copies of the UGT2B17 gene or they lack one copy, then they will excrete less testosterone than an individual that has two copies of the gene.
The amount of testosterone excreted varies between individuals. The average T/E ratio for Koreans was 0.15 and the average T/E ratio for Swedes was 1.8. Their genes can explain the differences. 67.7% of Koreans lacked two copies of the UGT2B17 gene (del/del genotype) and 22.7% lacked one copy of the UGT2B17 gene (del/ins genotype). This gene codes for the UGT2B17 enzyme responsible for excretion of testosterone in the urine. Note: enzymes called sulphate transferases are also involved in the elimination of testosterone which explains the small amount of testosterone excreted by people lacking two copies of the UGT2B17 gene.
What this means is that a person with a naturally low ratio of testosterone to epitestosterone in their urine can inject testosterone and not exceed the 4:1 ratio and their urine will not be tested using the carbon isotope test. Meaning they can inject testosterone right before a competition and get away with it.
Another problem is that athletes can microdose testosterone. In one study 9 people took 3.5mg of testosterone enanathate per kg of body weight once per week for 6 weeks. For someone who weighs 100kg that is almost 350mg of testosterone, they showed this amount of testosterone to be performance enhancing. The results show that 4/9 of the people taking this amount did not exceed the 4:1 ratio of testosterone to epitestosterone. So if these people were tested under WADA’s current test for testosterone they would pass the test. 44% of the people in the study can get away with taking a performance enhancing dose of testosterone enanathate year round and never get caught.
This also isn’t taking into account the different esters available of testosterone. The testosterone used in the study was testosterone enanathate which is testosterone with a 7 carbon ester chain, the addition of an ester chain increases the half-life. Athletes can use shorter acting versions of testosterone such as testosterone propionate, which has a 3 carbon ester chain. A popular PED for athletes is TNE (testosterone no ester), this is testosterone with no ester attached. TNE is short acting and with careful dosing athletes can use testosterone right up to competition. Other popular preparations are testosterone gels and patches which are short acting.
Two common preparations of testosterone available to athletes. The longer the ester chain the longer the half life.
Another method to beat the doping tests is by injecting epitestosterone along with testostereone. Jenapharm a pharmacuetical company founded in East Germany produced epitestosterone propionate which was exclusively used by the doping system since it had no commercial value. Injections of epitestosterone propionate and testosterone propionate were given to top level athletes to bring the T/E ratio back into the normal range.
To tackle the problems with the T/E ratio test, WADA came out with the biological passport. Using an individual’s T/E ratio as a reference is much more reliable than using the 4:1 T/E threshold. By measuring the T/E ratio (and other markers) multiple times through time and using statistics, an individual reference range is created. If a new measurement goes outside this reference range the athlete is suspected of doping. This means someone who lacks two copies of the UGT2B17 gene (meaning a naturally low T/E ratio) can get caught.
The way athletes get around this test is by microdosing or carefully dosing their testosterone around doping tests. By microdosing, the T/E ratio will not increase significantly and it will only raise for a short period. If an athlete carefully doses TNE their T/E ratio can return to normal within a few hours. Also, the biological passport only becomes effective when multiple data points are made (measurements of biological markers). This presents a problem for athletes who have never been tested.
Despite WADA’s efforts and over a billion dollars put into anti-doping, they still cannot catch every athlete who decides to take testosterone, a PED created almost a century ago.