September 05, 2006
Tuesday

Gretchen Wilson's country music smash hit, "All Jacked Up", wasn't exactly the most ladylike song to take-off from Nashville's Music Row last year. This isn't surprising considering that previously she became famous for confessing to be a "Redneck Woman."

But there may even be a chemical explanation for Ms. Wilson's affinity for mud, trucks and guns, if Floyd Landis's explanation for his failed urine test holds water, so to speak.

His urine sample was routinely taken after the 17th stage of the Tour de France this year, where Mr. Landis mounted an impressive come-from-behind effort in the Alps. After two days of high, but declining performance, he narrowly gave up the lead. More importantly, however, he retook the yellow jersey (which represents best standing overall) and wore it all the way to the Arc de Triumph where a champion's reception awaited - along with a team of analytical chemists.

Failing to keep up during the 16th stage of the tour, then surging to the fore during the 17th, there was bound to be speculation about doping in between. Chemical testing of two urine samples that were collected after his peak performance, using multiple protocols that are designed to thwart even biochemically sophisticated cheats, consistently pointed to abnormal levels of the male hormone testosterone.

But what constitutes "abnormal"? If a certain politician is to be believed, there are girlie-men and manly-men in the government of California. This may be true, but it is more likely a result of naturally varying levels of testosterone than party affiliation.

Instead of simply setting an arbitrary limit for testosterone, which might lead to more false positive tests of manly-man cyclists, the key result is the ratio of testosterone to epitestosterone. Both associated with the development of male characteristics, the effects of these two hormones counteract one another, so their natural ratio is usually very close to one - regardless of manliness.

This ratio can fluctuate naturally, but not very much. A T/E ratio greater than 4 suggests someone is trying to fool Mother Nature. It's a lot harder to fool a GC-mass spec, however.

The "GC" part of this ubiquitous instrument in chemistry labs - whether used in forensics or pure research - stands for gas chromatography, which is just a way of separating the multitude of chemical compounds in a complex liquid mixture, such as urine. In essence, the different molecular structures get carried along at different speeds by the carrier gas, usually an inert gas such as helium.

The "chromatography" part refers to the historical, and colorful, discovery of this general technique for separating molecules according to their average flow rate through some uniform medium. In the Tour de France, the cyclist with the overall fastest pace wears a yellow jersey, but in chromatography, the fastest compound can be any color, or even colorless. We say that it has the shortest "retention time".

Long retention times indicate that the compound has significant interactions with the stationary medium, but it is difficult to be more specific than that. This is where the "mass spec" part comes in. Mass spectroscopy refers to one of many methods of weighing molecules. Note that while jockeys get weighed before a race, molecules get weighed after - since "the race" is an analytical chemist's way of separating the molecules.

After his test results came in, Mr. Landis suggested that his elevated testosterone levels might have had any number of innocent causes. For instance, he said that he had taken a few shots of Jack Daniel's whiskey as a way to relax after his dismal performance in stage 16. He then offered a vague recollection of an article that suggested a rise in testosterone levels after consumption of such beverages. If this research holds up, I am sure that the good folks in Lynchburg, Tennessee, will get right on the job of promoting it.

It wouldn't help Mr. Landis though, since the mass spec data don't just indicate the amount of testosterone, they also point to possible sources, while all but ruling-out others. This is because while all testosterone molecules have exactly the same molecular structure, they don't all weigh the same.

Most carbon atoms have an atomic weight of 12, but roughly one percent weigh-in at 13. There are only 19 carbons in a testosterone molecule (along with 28 hydrogens and a pair of oxygens in this smallest unit androgenic bonding), so this mass difference shows up easily. The so-called "isotopic ratio" of carbon-12 to carbon-13 in the many, many molecular masses of the testosterone component of a urine sample can point to whether the hormone was made in a human body, or a pharmaceutical laboratory.

This is because our adrenal glands make hormones differently than pharmaceutical companies do. Not just in scale, either. Using a team of enzymes, our bodies chemically modify cholesterol to make testosterone and other hormones, including epitestosterone. To make testosterone cheaply, pharmaceutical companies use synthetic reagents to modify an isolate from soy.

Both cholesterol and the soy isolate are in turn made from simpler carbon building blocks. Depending on which enzymes are used in the natural synthetic steps, and where the carbon building blocks came from, the isotopic ratios will differ slightly. There is very little wiggle room for an athlete that gets busted by an isotopic fingerprint.

There is always some uncertainty in a scientific result, however. Perhaps he had a heap of tofu, more than a few Jack chasers, and then an enzymatic miracle occurred?

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More Chemical Eye On... by Preston MacDougall

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