Alleged blood doping and Lance Armstrong: Blood Doping and False Negatives

My apologies to my occasional readers, as I've been on hiatus for a few months now. Between travel to a conference in Japan, sporadic progress in my research, and a dearth of other things that have come up, I've had little time or inclination to update this blog.

Now that things have settled down, I aim to both put up some more interesting posts in the coming weeks and months, and to also go through the backlog of updates that I'd planned and penned, but had not yet posted.

The New York Times has released an interesting article regarding Lance Armstrong and his alleged doping, and follows a release by the CEO of the United States Anti-Doping Agency (USADA) discussing the findings of their recent investigation. Though I am only vaguely aware of the recent scandal surrounding Armstrong, I thought it a worthwhile piece to share, and to offer some comments along the way.

Firstly, a quick look through the NYT article indicates that new tests on blood samples obtained for the purposes of drug testing were anomalous, and were consistent with blood doping. In my work and research, the main method of concealing the use of illicit, recreational or performance enhancing drugs is through urine adulteration. Indeed, this method is arguably rather simple, and can be extremely effective if one uses the correct chemicals in the correct volumes and concentrations, and is able to adulterate the urine unnoticed.

Blood ≠ Urine

Such a method of concealing alleged drug use does not translate well to a biological matrix such as blood, and for a range of reasons:

1. Addition of adulterants to blood is likely to have a significant effect on the chemical composition of the blood, and will potentially affect the settling of red blood cells. Consequently, it would potentially have a visible effect on the blood sample.
2. For obvious reasons, it would be difficult and quite dangerous to attempt to adulterate the blood prior to sampling. 
3. More importantly, it would be unlikely that a person attempting to adulterate their blood would be able to do so during sampling. Indeed, it would be more likely for a co-conspirator working in a drug testing laboratory to accomplish this.

However, has not claimed that this type of adulteration has occurred. Rather, it has been alleged that a doctor associated with Armstrong administered saline solution to the athlete, thereby restoring normal blood levels. I'm aware of two main methods of blood doping where such a strategy would be effective:

1. Instances where blood is collected and stored, and prior to an event, the stored blood is infused into the athlete's bloodstream, thereby increasing the number of red blood cells. This in turn improves the oxygen-carrying capacity of the blood, and ultimately improves an athlete's performance and endurance. Injecting saline prior to a blood test will reduce the concentration/count of red blood cells to a normal value, and will potentially provide a false negative for blood doping.
2. Injection of performance-enhancing substances like erythropoietin (EPO) will potentially be detectable in blood. Similar to the point above, transfusing "normal" (unadulterated) blood, will "hide" traces of performance enhancing compounds.

It would appear that USADA alleges that Armstrong and his cycling colleagues used EPO, testosterone and other drugs to enhance his performance, and that transfusion of stored, "normal" blood was used to mask this. This technique was helped by the fact that previous methods used for testing EPO levels were not necessarily sensitive or specific, and as such, could be fooled. On this point, the press release from USADA indicates that:

"The evidence shows beyond any doubt that the US Postal Service Pro Cycling Team ran the most sophisticated, professionalized and successful doping program that sport has ever seen."

Again, though I am only partially aware of this case, its ramifications, and have only read part-way through USADA's document "Reasoned Decision of the United States Anti-Doping Agency on Disqualification and Ineligibility; United States Anti-Doping Agency v. Lance Armstrong", this entire debacle raises some interesting questions regarding the use of performance enhancing drugs, and the methods that are currently used to detect doping.

Certainly, it is unfair that some athletes will use, or attempt to use, performance enhancing substances, and that it is a sad example of poor sportsmanship, and should not be condoned in any way, shape or form. Indeed, significant advances in detecting blood doping and urine adulteration have been made in recent years, and improvements, refinements and new techniques are continually being explored. It therefore raises the questions: "Will testing agencies be able to keep up with designer drugs and the people who use them?" and "Will there come a point where it is no longer possible or practical to engage in doping?".

I cannot definitively answer either question, but I have found it interesting talking with colleagues about such matters. Opinion appears to be firmly split. On one hand, many of my peers feel that it is only a matter of time before the flow of novel designer drugs slows, and that testing agencies will gain the upper hand. Others are more cynical, and feel that toxicologists and testing agencies will always be playing a game of "catch-up", never truly able to be in a position where all novel drugs can be detected in a safe, effective, sensitive and specific manner.

I guess only time will tell...

Article published in the TIAFT Bulletin

More good news coming from my recent trip to Hamamatsu, Japan, for the 50th annual meeting for The International Association of Forensic Toxicologists (TIAFT). Aside from having the honour of travelling to Japan for this conference, which was most informative and allowed me to meet with my peers, I was given the opportunity to submit my research to the TIAFT Bulletin.

I was recently informed that my article, titled "Effect of selected oxidising agents on the detection of 11-nor-9-carboxy-delta-9-tetrahydrocannibinol (THC-COOH) in spiked urine" was accepted and published in the 42nd issue of the TIAFT Bulletin.

Abstract from my article. A link to the full issue of the TIAFT Bulletin and my article is included below.

As such, this is a quick post to provide a link to the latest issue of the TIAFT Bulletin (at the time of writing), and more specifically, a link to my article.

Link to a .pdf of the article in question.

Until next time,
Nathan

Animals and drugs don't mix: Dolphins and Buprenorphine

Another sad article that I came across whilst at work today concerning the deaths of two dolphins at a marine park in Switzerland.

A nuclear submarine dolphin

Housed at the Connlyland Marine Park in Lipperswil, Switzerland, the two dolphins died in November of 2011. Some days prior to the deaths, bosses at the zoo allegedly rented out an area of the marine park, adjacent to the dolphin training pool, to a weekend-long rave.

Initially, there were reports that the dolphins had died as a result of the loud music from the party, which was thought to have damaged their sensitive auditory structures, hindering their hearing and sonar ability. Indeed, local animal rights groups recorded sound levels outside of the park during the rave, and apparently recorded sound levels at over 100 decibels. However, Swiss prosecutors instead considered levelling charges of negligence against personnel at the zoo, as they considered the possibility that the deaths had been due to improper administration of antibiotics.

A recently leaked toxicology report has, however, possibly shed new light on the deaths of the dolphins, known as Shadow and Chelmers. Apparently, significant quantities of buprenorphine, a semi-synthetic opioid, were found in the dolphins' urine. As an aside, the toxicologist in me,feels that it should be pointed out that, as buprenorphine undergoes significant first-pass metabolism, it is more likely that they found nobuprenorphine in the urine samples submitted for analysis.

It would then appear that the deaths of the dolphins was instead due to an overdose of buprenorphine.

Buprenorphine: not dolphin-friendly...

As mentioned above, buprenorphine is a semi-synthetic opioid that has a range of clinical uses, including the treatment of opiate addiction and mild to chronic pain. It is, however, also being increasingly used as a recreational drug, and administered via insufflation. This unfortunately raises the possibility that one or more party-goers present at the rave may have administered buprenorphine to the dolphins.

According to one of the zookeepers at the marine park, Nadja Gasser, the dolphins suffered a horrific and slow death:

“He was drifting under the water and was clearly in trouble and so we jumped into the water. We tried to hold him. He was shaking all over and was foaming at the mouth... The death went on for over an hour. It was horrendous.”

Dutch marine biologist, Cornelis van Elk, commented that opiates are highly dangerous to aquatic mammals:

"The reason is that dolphins are conscious breathers, which means they actively decide when to come to the surface to breathe... Drugging them with opiates causes this part of the brain to switch off, with fatal consequences."

An editorial by Elizabeth Batt, from Digital Journal, makes a rather astute comment regarding the unfortunate deaths of these animals:

"At the end of the day, it matters not whether the dolphins died from environmental noise, antibiotics or as it now seems likely, a heroin substitute. They died from negligence at the top level. Despite the warnings and the concerns... park bosses and Veterinary Officials went ahead and allowed the rave anyway. And two dolphins at Connyland Zoo suffered indescribable painful deaths..."

It is truly a pity that events unfolded in the manner that they did. It seems unbelievable that a zoo/marine park would approve to have a rave next to the exhibit next to any animal, and to not have any protections in place to prevent stupid/inebriated/intoxicated reveller/s from interfering with the animals seems downright bizarre... Well, let us hope that this doesn't happen again.

Until next time,

Nathan

A Sad Story, or Why Oleander and Giraffes Don't Mix

My partner recently forwarded me a news story from Tucson, Arizona, regarding the unfortunate death of a giraffe at the Reid Park Zoo.

Based on the article, it would appear that a giraffe was accidentally fed brush trimmings that included leaves from the ornamental plant Oleander (Nerium Oleander). Oleander is a rather common ornamental plant, and is indeed ubiquitous in and around Sydney. Indeed, my primary school in South-West Sydney has a large oleander tree in the rear playground.  The leaves bear a superficial resemblance to those of the olive plant (hence the name), and have rather pretty white/pink flowers. It is rather hardy as well, being drought tolerant and capable of withstanding mild frosts, and hence has found its niche as an ornamental plant.

Pretty. Also deadly. Pretty deadly, I suppose?

Sadly, oleander is also incredibly toxic, with the leaves, bark and sap all containing a class of compounds known as cadiac glycosides. For the giraffe in the above article, what can only be presumed as an innocent mistake by a zookeeper was ultimately fatal. The response of zoo officials, in "carefully considering removing all the Oleander that's surrounding the outside of the park" is understandable.

As mentioned, oleander contains cardiac glycosides, which include the chemical oleandrin (structure below):

Definitely not giraffe friendly...

Despite the above case, cardiac glycosides are useful chemicals, and in a medical setting are used to treat congestive heart failure and arrhythmia. This use is mediated by inhibition of the sodium-potassium ion pumps present in cell membranes, which ultimately leads to an increased intracellular concentration of calcium. This, in turn, improves the force of contraction by cardiac muscle, improving cardiac output.

Unfortunately, compounds like oleandrin can also be insidiously toxic, with high doses capable of decreasing cardiac function and reducing the availability of oxygen to the tissues of the body. Without treatment, this can most certainly be fatal. Multiple cases of deaths due to ingestion (accidental or otherwise) ingestion of oleander and preparations of its leaves.  In 2000, two toddlers, aged two and three years old, died as a result of ingesting the leaves. This case is in itself unusual, as the leaves are reported to have an incredibly bitter taste, with Dr. Clarke, a medical toxicologist and director of the California Poison Control Centre, commenting that "There is not a single other case in the American literature that I know of of people eating oleander leaves and dying...".

Similarly, in the Annals of Emergency Medicine, it is reported that in 1985, a woman died after drinking a herbal tea prepared from the leaves of Neruim oleander. The article itself is interesting, as it is alleged that the woman mistook the leaves of the oleander plant for those of eucalyptus. This mistake proved fatal, as within ten hours the woman was displaying symptoms of confusion and impaired cardiac function. Staff at the hospital identified the leaves as those of the oleander plant, and took appropriate measures to prevent death. However:

"...despite these measures... the patient's cardiac rhythm deteriorated to an agonal rhythm and then to asystole. A transthoracic pacemaker was inserted... but no ventricular capture or palpable pulse resulted, and the patient was pronounced dead"

Treatment options are available in cases of oleander poisoning. Often, anti-digoxin Fab is given, which is the anti-digoxin antibody fragment. This antibody is routinely given for digoxin poisoning, which is another cardiac glycoside found in Digitalis.

Deaths due to oleander poisoning are not only limited to humans and giraffes. From livestock, including cattle and horses, through to domestic animals including dogs, oleander represents a real danger. Even goats, reputed by an old wive's tale to eat almost anything, are not immune. In general, it would appear that people are not aware of the toxicity of plants, oleander or otherwise.

As a sidenote, I feel that it would be remiss of me not to note one very interesting area of research concerning oleandrin. In the past few years, there has been some promising research indicating the possibility that oleandrin may be an anti-cancer agent. It has been found that oleandrin mediates apoptosis in tumour cells, but not in primary cells. If true, this would indicate that oleandrin may work as an anti-cancer agent with minimal side effects at the required dose. In vitro studies have also demonstrated that oleandrin may aid in the treatment of a wide range of cancers, including leukemia, pancreatic cancer and prostate cancer.

That being said, this research is still only in its early stages. However, I will be cautiously waiting for further developments in this area. This should not be an excuse for anyone to attempt ingestion of oleander. I trust that such a disclaimer is not warranted, but just to be safe, I shall include it again:

Please don't eat/ingest oleander. It really isn't a good idea. I promise. 

Until next time,
Nathan

The War on Drugs - Press Release by Australia21

In recent days there has been a really fascinating media release from a think tank known as Australia 21. Described on their website as "... an independent, non-profit organisation whose core purpose is multidisciplinary research and inquiry on issues of strategic importance to Australia in the 21st century", Australia 21 has released their much-anticipated report on discussing the costs and benefits of any potential changes to Australia's policy on illicit drugs.

The report itself is rather controversial. Not only does it broach a subject that many people still find taboo in this day and age, it also has quite a controversial title: "The Prohibition of Illicit Drugs is Killing and Criminalising Our Children and We Are All Letting It Happen". Title notwithstanding, the report makes for fascinating reading, though I must admit that at the time of writing, I am only part way through reading it. What has been clear so far is that the release of this report has already lead to debate within the wider community over current legislation regarding illicit/recreational drugs. As mentioned in a previous post, this is a contentious issue that, unfortunately, tends to be political suicide for those willing to bring it up for discussion and debate.

At any rate, the fact that drug legalisation and drug policy is once again being discussed does give one hope that a more reasonable and evidence-based approach to recreational drugs, harm minimisation and harm reduction will be reached.

Until next time,

Nathan

2012 TIAFT Conference

Earlier this year I submitted an abstract to the chairperson of the 2012 organising committee of The International Association of Forensic Toxicologists (TIAFT), with the hopes of attending the upcoming conference in Hamamatsu, Japan.

Logo for the conference I will have the privilege of attending

Back in 2009 I was afforded the opportunity to attend the TIAFT conference in Geneva, Switzerland. It was truly amazing to meet my peers, and to immerse myself in the buzzing atmosphere of toxicologists sharing their latest research, curious cases and other bits and pieces of fascinating information.

At any rate, I received word today that I will be attending the 50th annual meeting of The International Association of Forensic Toxicologists, and will be presenting a poster at this conference. The abstract I submitted was titled "Further study of the detection of oxidation products of 11-nor-9-carboxy THC (THC-COOH) following urinary adulteration", and will discuss the effects of different oxidising adulterants on the detection of a key secondary metabolite of delta-9-tetrahydrocannibinol (THC) in urine. I really haven't mentioned this research prior to this post, so at some point in the future I shall provide a quick intro into what I am currently researching.

In the coming weeks I will provide a preview of my poster. More importantly, I need to finalise my research so I have a poster to present!

The coming weeks shall be busy indeed.

With kindest regards,
Nathan

Artificial Sweeteners - Aspartame

A few weeks back I had the privilege to go on a road trip to Batemans Bay with some of my friends. It was a wonderful break from work, and allowed me to catch up with friends that, due to distance and/or conflicting schedules, I may only see a couple of times per year.

One morning, we were having breakfast, in preparation for a big day. Sitting at a big table, we were eating, drinking and generally being merry. During breakfast, one of my friends leant over towards me and gestured towards the diet soda that I was drinking, and began the following exchange:

Friend: "Hey, you shouldn't drink that!"
Me: "Yeah, it is a bit early in the day for soft drink, but I don't feel like coffee or juice this morning..."
Friend: "No, you shouldn't drink that. Didn't you know that the fake sugar in that was banned in the UK? It causes cancer!"
Me: "Really? I'd wager that a company can't distribute a product with a known carcinogen, and not be the subject of a law suit."
Friend: "No, it's true! I read it somewhere..."

"I read it somewhere"... A line that always makes me cringe inwardly. Perhaps it is the scientist in me, but I do prefer a good reference as opposed to an anonymous source.

At any rate, just what was my friend referring to? The nods of assent he received from my other friends from around the dining table made it seem that this substance was well known. Well, it would appear that based on my choice of beverage that fateful morning, it seems likely he was referring to aspartame, an artificial sweetener used as a sugar substitute.

Oh how sweet it is... Approximately 200 times sweeter than sucrose, in fact!

So, why does the above molecule have such a bad reputation? Well, aspartame was discovered way back in 1965 by a chemist working for G.D. Searle & Company. Originally synthesised in order to produce a potential antiulcer drug, the sweetness of aspartame was discovered when a chemist licked his finger in order to pick up a piece of paper. Evidently this chemist was not wearing gloves at the time, as during the aforementioned synthesis, his finger had been contaminated by aspartame.

Shortly after approval by the Food and Drug Administration, a chain letter posted under the pseudonym of Nancy Markle, warning of the inherent risks posed by aspartame. The alleged dangers of aspartame included an increased incidence of multiple sclerosis and brain tumors, and a dire warning that when exposed to heat, that aspartame decomposes to produce methanol, and hence consumption of products containing aspartame could lead to methanol poisoning.

Indeed, there have been numerous claims regarding aspartame and its relative safety for human consumption. The safety of aspartame has been studied extensively, and has repeatedly been deemed safe for human consumption. Regarding intake and consumption, the FDA has set the acceptable daily intake (ADI) of aspartame at 50 mg/kg of body weight. To put this into perspective, a 350-375 mL diet soft drink may contain around 180 milligrams of aspartame. For a 75 kg individual, it would therefore take approximately 20 cans of diet soft drink to exceed the ADI of aspartame.

One of the other claims made against consumption of aspartame is that it breaks down in the body to form highly toxic compounds, including aspartic acid, phenylalanine and methanol.

•  Aspartic acid: One of the most commonly encountered amino acid in a regular diet. A rather fascinating clinical study by Magnuson et. al. (2007), however, found no evidence of neurotoxic effects derived from consumption of aspartic acid. 
• Phenylalanine: One of the essential amino acids, one is likely to consume far more phenylalanine during their regular diet than from diet soft drink. That being said, those with phenylketonuria are advised to abstain from food products that contain significant amounts of phenylalanine or aspartame. 
• Methanol: The metabolism of methanol to formaldehyde, and then formaldehyde to formic acid, is implicated as one of the dangers of aspartame use. It should be noted, however, that the amount of methanol absorbed by the body as a result of consuming aspartame-containing products, is less than what would be absorbed if one consumed various fruit juices or fermented beverages. Indeed, in the article by Magnuson et. al. (above), it was found that consuming the maximum expected amount of aspartame did not result in elevated blood levels of methanol, formaldehyde or formic acid.

As for claims regarding aspartame's alleged ability to cause brain cancer, no significant links have been found to date. One study by European Ramazzini Foundation of Oncology and Environmental Sciences (ERF) alleged that aspartame is a carcinogen at normal dietary doses. However, this study has been discredited, and in fact, the European Food Safety Authority evaluated this study back in 2010. This evaluation found that there were multiple significant design flaws in the study by the ERF, and that as such, there was insufficient evidence to suggest that the safety of aspartame needed to be reconsidered.

Claims have also been made that have alleged that consumption of aspartame may result in negative neurological and psychiatric symptoms, including seizures, headaches and mood swings. Lajtha et. al. (1993) found that with regards to the biochemistry of aspartame, there is no evidence that aspartame could conceivably lead to neurotoxic effects.

So there you have it. One artificial sweetener and a number of concerns. Though it would not surprise me that in the future the safety of aspartame will be reviewed once again, for the moment it is crucial to note that there is no evidence to suggest that the use of aspartame represents any real cause for concern.

Now where did I put that can of soda...?