Archive for the ‘Antidoping’ Category

Research news from Journal of Mass Spectrometry

A new mass spectrometry test can help sports anti-drug doping officials to detect whether an athlete has used drugs that boost naturally occurring steroid levels. The test is more sensitive compared to previous alternatives, more capable of revealing specific suspicious chemical in the body, faster to perform, and could be run on standard drug-screening laboratory equipment. The new test is announced in a special issue of the Journal of Mass Spectrometry that concentrates on detecting drugs in sports.

One of the roles of the masculinising hormone testosterone is to increase muscle size and strength. Taking extra testosterone, or taking a chemical that the body can use to create extra testosterone, could therefore enhance an athlete’s performance. For this reason taking it is banned by the World Anti-Doping Agency (WADA).

The exact level of testosterone varies considerably between different people, so simply measuring total testosterone in an athlete’s urine can not show whether he or she has deliberately taken extra. There is, however, a second chemical in the body, epitestosterone, which is normally present in approximately equal proportions to testosterone. Comparing the ratio of testosterone to epitestosterone can then indicate whether testosterone or a precursor has been taken.

The problem is that it is not always easy to measure these two substances, particularly as they are only present in urine at very low concentrations.

A team of scientists the Sports Medicine Research and Testing Laboratory at the University of Utah have developed a test that makes use of liquid chromatography-tandem mass spectrometry. This method has incredibly high sensitivity (down to 1 ng/ml) and increases the power with which officials can search for both testosterone and epitestosterone within a sample.

“Our system means that we can determine the testosterone/epitestosterone ratio in a sample with greater confidence, and therefore be in a better position to spot doping violations without falsely accusing innocent athletes,” says lead investigator Dr Jonathan Danaceau.

“Not only is the test more sensitive, it is also faster to perform,” says colleague Scott Morrison.

“Having this sort of test available makes cheating harder and lets us take one more step towards enabling free and fair competition,” says Laboratory Director Dr Matthew Slawson.

This paper is part of a special issue for the Olympic Games from the Journal of Mass Spectrometry which focuses of drug use in sport. The issue is available free of charge online for one month at http://www.interscience.wiley.com/journal/jms. The other articles publishing in this issue are:

 

  • History of Mass Spectrometry at Olympic Games (DOI: 10.1002/jms.1445)
  • Nutritional supplements cross-contaminated and faked with doping substances (DOI: 10.1002/jms.1452)
  • Hair analysis of anabolic steroids in connection with doping control results from horse samples (DOI: 10.1002/jms.1446)
  • Mass spectrometric determination of Gonadotrophin releasing hormone (GnRH) in human urine for doping control purposes by means of LC-ESI-MS/MS (DOI: 10.1002/jms.1438)
  • Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and inter-laboratory comparison (DOI: 10.1002/jms.1434)
  • Mass Spectrometry of Selective Androgen Receptor Modulators (DOI: 10.1002/jms.1438)
  • Can glycans unveil the origin of glycoprotein hormones? – human chorionic gonadotropin as an example (DOI: 10.1002/jms.1448)
  • A High-Throughput Multicomponent Screening Method for Diuretics, Masking Agents, Central Nervous System Stimulants and Opiates in Human Urine by UPLC-MS/MS (DOI: 10.1002/jms.1436)
  • The application of carbon isotope ratio mass spectrometry to doping control (DOI: 10.1002/jms.1437)
  • Identification of zinc-alpha-2-glycoprotein binding to clone ae7a5 anti-human epo antibody by means of nano-hplc and high-resolution highmass accuracy esi-ms/ms (DOI: 10.1002/jms.1444)
  • Low LC-MS/MS Detection of Glycopeptides Released from pmol Levels of Recombinant Erythropoietin using Nanoflow HPLC-Chip Electrospray Ionization (DOI: 10.1002/jms.1439)
  • Introduction of HPLC/Orbitrap mass spectrometry as screening method for doping control (DOI: 10.1002/jms.1447)

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Article adapted by MD Sports from original press release.
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Contact: Jennifer Beal
Wiley-Blackwell

And it increases endurance to run a mile and decreases inflammation

The Salk Institute scientist who earlier discovered that enhancing the function of a single protein produced a mouse with an innate resistance to weight gain and the ability to run a mile without stopping has found new evidence that this protein and a related protein play central roles in the body’s complex journey to obesity and offer a new and specific metabolic approach to the treatment of obesity related disease such as Syndrome X (insulin resistance, hyperlipidemia and atherosclerosis).

Dr. Ronald M. Evans, a Howard Hughes Medical Investigator at The Salk Institute’s Gene Expression Laboratory, presented two new studies (date) at Experimental Biology 2005 in the scientific sessions of the American Society for Biochemistry and Molecular Biology. The studies focus on genes for two of the nuclear hormone receptors that control broad aspects of body physiology, including serving as molecular sensors for numerous fat soluble hormones, Vitamins A and D, and dietary lipids.

The first study focuses on the gene for PPARd, a master regulator that controls the ability of cells to burn fat. When the “delta switch” is turned on in adipose tissue, local metabolism is activated resulting in increased calorie burning. Increasing PPARd activity in muscle produces the “marathon mouse,” characterized by super-ability for long distance running. Marathon mice contain altered muscle composition, which doubles its physical endurance, enabling it to run an hour longer than a normal mouse. Marathon mice contain increased levels of slow twitch (type I) muscle fiber, which confers innate resistance to weight gain, even in the absence of exercise.

Additional work to be reported at Experimental Biology looks at another characteristic of PPARd: its role as a major regulator of inflammation. Coronary artery lesions or atherosclerosis are thought to be sites of inflammation. Dr. Evans found that activation of PPARd suppresses the inflammatory response in the artery, dramatically slowing down lesion progression. Combining the results of this new study with the original “marathon mouse” findings suggests that PPARd drugs could be effective in controlling atherosclerosis by limiting inflammation and at the same time promoting improved physical performance.

Dr. Evans says he is very excited about the therapeutic possibilities related to activation of the PPARd gene. He believes athletes, especially marathon runners, naturally change their muscle fibers in the same way as seen in the genetically engineered mice, increasing levels of fat-burning muscle fibers and thus building a type of metabolic ‘shield” that keeps them from gaining weight even when they are not exercising.

But athletes do it through long periods of intensive training, an approach unavailable to patients whose weight or medical problems prevent them from exercise. Dr. Evans believes activating the PPARd pathway with drugs (one such experimental drug already is in development to treat people with lipid metabolism) or genetic engineering would help enhance muscle strength, combat obesity, and protect against diabetes in these patients.

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Article adapted by MD Sports Weblog from original press release.
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Contact: Sarah Goodwin
Federation of American Societies for Experimental Biology

When given extra shots of the plant steroid brassinolide, plants “pump up” like major league baseball players do on steroids. Tracing brassinolide’s signal deep into the cell’s nucleus, researchers at the Salk Institute for Biological Studies have unraveled how the growth-boosting hormone accomplishes its job at the molecular level.The Salk researchers, led by Joanne Chory, a professor in the Plant Molecular and Cellular Biology Laboratory and a Howard Hughes Medical Institute investigator, published their findings in this week’s journal Nature.

“The steroid hormone brassinolide is central to plants’ growth. Without it, plants remain extreme dwarfs. If we are going to understand how plants grow, we need to understand the response pathway to this hormone,” says Chory. “This study clarifies what’s going on downstream in the nucleus when brassinolide signals a plant cell to grow.”

Brassinolide, a member of a family of plant hormones known as brassinosteroids, is a key element of plants’ response to light, enabling them to adjust growth to reach light or strengthen stems. Exploiting its potent growth-promoting properties could increase crop yields or enable growers to make plants more resistant to drought, pathogens, and cold weather.

Unfortunately, synthesizing brassinosteroids in the lab is complicated and expensive. But understanding how plant steroids work at the molecular level may one day lead to cheap and simple ways to bulk up crop harvests.

Likewise, since low brassinolide levels are associated with dwarfism, manipulating hormone levels during dormant seasons may allow growers to control the height of grasses, trees or other plants, thereby eliminating the need to constantly manicure gardens.

Based on earlier studies, the Salk researchers had developed a model that explained what happens inside a plant cell when brassinolide signals a plant cell to start growing.

But a model is just a model. Often evidence in favor of a particular model is indirect and could support multiple models. Describing the components of the signaling cascade that relays brassinolide’s message into a cell’s nucleus, postdoctoral researcher and lead author of the study Grégory Vert, now at the Centre national de la recherche scientifique (CNRS) in Montpellier, France, said, “All the players are old acquaintances and we knew from genetic studies that they were involved in this pathway. But when we revisited the old crew it became clear that we had to revise the original model.”

When brassinosteroids bind a receptor on the cell’s surface, an intracellular enzyme called BIN2 is inactivated by an unknown mechanism. Previously, investigators thought that inactivation of BIN2, which is a kinase, freed a second protein known as BES1 from entrapment in the cytoplasm, the watery compartment surrounding a cell’s nucleus, and allowed it to migrate or “shuttle” into the nucleus where it tweaked the activity of genes regulating plant growth.

A closer inspection, however, revealed that BIN2 resides in multiple compartments of a cell, including the nucleus, and it is there–not in the cytoplasm–that BIN2 meets up with BES1 and prevents it from activating growth genes. “All of a sudden the ‘BES1 shuttle model’ no longer made sense,” says Vert, adding that it took many carefully designed experiments to convince himself and others that it was time to retire the old model.

A new picture of how brassinosteroids stimulate plant growth now emerges based on those experiments: steroid hormones are still thought to inactivate BIN2 and reciprocally activate BES1, but instead of freeing BES1 to shuttle into the nucleus, it is now clear that the crucial activation step occurs in the nucleus where BES1 is already poised for action. Once released from BIN2 inhibition, BES1 associates with itself and other regulatory factors, and this modified form of BES1 binds to DNA, activating scores of target genes.

Referring to the work of Vert and other members of the brassinosteroid team, Chory says, “The old model may be out, but Greg’s new studies, together with those of former postdocs, Yanhai Yin and Zhiyong Wang, have allowed us to unravel the nuclear events controlling brassinosteroid responses at the genomic level. This turns our attention to the last mystery: the gap in our understanding of the events between steroid binding at the cell surface and these nuclear mechanisms.”

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Article adapted by MD Sports Weblog from original press release.
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Contact: Gina Kirchweger
Salk Institute

The Salk Institute for Biological Studies in La Jolla, California, is an independent nonprofit organization dedicated to fundamental discoveries in the life sciences, the improvement of human health and the training of future generations of researchers. Jonas Salk, M.D., whose polio vaccine all but eradicated the crippling disease poliomyelitis in 1955, opened the Institute in 1965 with a gift of land from the City of San Diego and the financial support of the March of Dimes.

The Johns Hopkins scientists who first created “mighty mice” have developed, with pharmaceutical company Wyeth and the biotechnology firm MetaMorphix, an agent that’s more effective at increasing muscle mass in mice than a related potential treatment for muscular dystrophy now in clinical trials.

The new agent is a version of a cellular docking point for the muscle-limiting protein myostatin. In mice, just two weekly injections of the new agent triggered a 60 percent increase in muscle size, the researchers report in the Proceedings of the National Academy of Sciences, published and available publicly through the journal’s website.

The researchers’ original mighty mice, created by knocking out the gene that codes for myostatin, grew muscles twice as big as normal mice. An antibody against myostatin now in clinical trials caused mice to develop muscles 25 percent larger than those of untreated mice after five weeks or more of treatment.

The researchers’ expectation is that blocking myostatin might help maintain critical muscle strength in people whose muscles are wasting due to diseases like muscular dystrophy or side effects from cancer treatment or AIDS.

“This new inhibitor of myostatin, known as ACVR2B, is very potent and gives very dramatic effects in the mice,” says Se-Jin Lee, M.D., Ph.D., a professor of molecular biology and genetics in Johns Hopkins’ Institute for Basic Biomedical Sciences. “Its effects were larger and faster than we’ve seen with any other agent, and they were even larger than we expected.”

ACVR2B is the business end of a cellular docking point for the myostatin protein, and it probably works in part by mopping up myostatin so it can’t exert its muscle-inhibiting influence. But the researchers’ experiments also show that the new agent’s extra potency stems from its ability to block more than just myostatin, says Lee.

“We don’t know how many other muscle-limiting proteins there may be or which ones they are,” says Lee, “but these experiments clearly show that myostatin is not the whole story.”

The evidence for other players came from experiments with mighty mice themselves. Because these mice don’t have any myostatin, any effects of injecting the new agent would come from its effects on other proteins, explains Lee. After five injections over four weeks, mighty mice injected with the new agent had muscles 24 percent larger than their counterparts that didn’t get the new agent.

“In some ways this was supposed to be a control experiment,” says Lee. “We weren’t really expecting to see an effect, let alone an effect that sizeable.”

In other experiments with normal female mice, weekly injections of the new agent provided the biggest effect on muscle growth after just two weeks at the highest dose given (50 milligrams per kilogram mouse weight). Depending on the muscle group analyzed, the treated mice’s muscles were bigger than untreated mice by 39 percent (the gastrocnemius [calf] muscle) to 61 percent (the triceps).

After just one week, mice given a fifth of that highest dose had muscles 16 percent to 25 percent bigger than untreated mice, depending on the muscle group analyzed, and mice treated with one injection a week for two, three or four weeks continued to gain muscle mass.

But although the new agent seems quite promising, its advantage in potency also requires extra caution. “We don’t know what else the new agent is affecting or whether those effects will turn out to be entirely beneficial,” says Lee.

Lee says they also are conducting experiments with the mice now to see whether the effect lasts after injections cease and whether it helps a mouse model of muscular dystrophy retain enough muscle strength to prolong life.

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Article adapted by MD Sports Weblog from original press release.
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Contact: Joanna Downer
Johns Hopkins Medical Institutions

The research was funded by grants from the National Institute of Child Health and Human Development and the National Cancer Institute and by funds from Wyeth Research and MetaMorphix Inc. The new agent was produced and first tested at Wyeth, and the inhibitor used in the current mouse studies was produced at MetaMorphix. All of the mouse studies described in this article and in the PNAS paper were conducted in Lee’s laboratory at Johns Hopkins.

Authors on the report are Se-Jin Lee and Suzanne Sebald of Johns Hopkins; Lori Reed of Wyeth Exploratory Drug Safety, and Monique Davies, Stefan Girgenrath, Mary Goad, Kathy Tomkinson, Jill Wright and Neil Wolfman of Wyeth Discovery Research; Christopher Barker, Gregory Ehrmantraut, James Holmstrom and Betty Trowell of MetaMorphix Canada; Barry Gertz, Man-Shiow Jiang, Li-fang Liang, Edwin Quattlebaum and Ronald Stotish of MetaMorphix, Beltsville, Md.; Martin Matzuk of Baylor College of Medicine; and En Li of Harvard Medical School.

Myostatin was licensed by The Johns Hopkins University to MetaMorphix and sublicensed to Wyeth. Lee is entitled to a share of sales royalty received by The Johns Hopkins University from sales of this factor. The Johns Hopkins University and Lee also own MetaMorphix stock, which is subject to certain restrictions under university policy. Lee is a paid consultant to MetaMorphix. The terms of these arrangements are being managed by the university in accordance with its conflict of interest policies.

On the Web: http://www.pnas.org/cgi/content/abstract/0505996102v1

Genetic research into athletic ability should be encouraged for its potential benefits in both sport and public health, a leading group of scientists meeting at the University of Bath said today. Genetic research into athletic ability should be encouraged for its potential benefits in both sport and public health, a leading group of scientists meeting at the University of Bath said today.

However, ethical concerns, such as whether seeking information about differences between ethnic groups could be perceived as racist research, need to be properly addressed, they warn.

Their recommendations are published in a ‘position stand’ on genetic research and testing launched at the British Association of Sport & Exercise Sciences annual meeting today.

They call for more genetic research in the sport and exercise sciences because of the anticipated benefits for public health, but want researchers to take a more active role in debating the implications of their work with the public.

“If a powerful muscle growth gene was identified, on the one hand this could help develop training programmes that increase muscle size and strength in athletes, but even more importantly the knowledge could be used to develop exercise programmes or drugs to combat muscle wasting in old age,” said Dr Alun Williams from Manchester Metropolitan University, one of the report’s authors.

“We, as scientists investigating genetics, acknowledge a public concern about some genetic research and we believe scientists need to engage in public in debates about the potential benefits of their research.

“Research into the athletic success of East African distance runners or sprinters of West African ancestry might be perceived as unethical.

“But understanding the limits of human exercise capacity in sport could lead to the development of treatments for a range of diseases like cancer and cardiovascular disease.”

The potential applications of genetic testing in sport and exercise also raise some ethical concerns, for example in identifying potential athletic ability before birth.

An Australian company already offers the first genetic performance test (for the ACTN3 gene) which has been linked to sprint and power performance.

The report authors are sceptical about whether this test is useful but anticipate that more advanced versions of these tests will be available in future.

“We are not yet at a point where we can identify a potential future Olympic champion from genetic tests but we may not be very far away,” said Dr Williams, who wrote the report with Drs Henning Wackerhage (Aberdeen University), Andy Miah (University of Paisley), Roger Harris (University of Chichester) and Hugh Montgomery (University College London).

They highlight two dangers of genetic performance tests. Firstly, genetic performance tests might later be linked to disease. For example, a muscle growth gene may later be linked to cancer growth.

“Not all people may want to know, while young that they are at increased risk of cancer by early middle age, but they might inadvertently become aware of that just because they had a ‘sport gene’ test,” said Dr Williams.

Secondly, genetic performance tests can be performed even before birth and this may lead to the selection of foetuses or to abortions based on athletic potential.

The report recommends genetic counselling and that the testing should be confined to mature individuals who fully understand the relevant issues.

Genetic tests might also be used to screen for health risks during sport such as genes that are linked to sudden cardiac death.

Genetic tests for sudden cardiac death are already available but the report recommends that such testing should not be enforced on athletes.

Problems with mandatory testing are highlighted by the case of the basketball player Eddy Curry, who had an irregular heart beat.

Curry was asked by his club, the Chicago Bulls, to perform a predictive genetic test for a heart condition. The athlete refused and was traded to the New York Knicks who did not make such a demand.

In future, genetic tests might be used to identify those that respond with the biggest drop in cholesterol, blood pressure or glucose to exercise.

In a personalised medicine approach, such tests could be used to select subjects for therapeutic exercise programmes but scientists are concerned that this might undermine the ‘exercise for all’ message that already seems difficult to get across to the public.

The authors say that genetic testing might also be used to detect gene doping, which may be a real threat by the time of the London Olympics in 2012, or to show that positive doping tests are the result of a genetic mutation in an athlete.

The report recommends that genetic testing should be used for anti-doping testing as long as the genetic samples are destroyed after testing.

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Article adapted by MD Sports Weblog from original press release.
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Contact: Andrew McLaughlin
University of Bath

Whippets are bred for speed and have been clocked at speeds approaching 40 miles per hour over a 200-yard racing course. Scientists at the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health (NIH), have now discovered a genetic mutation that helps to explain why some whippets run even faster than others. Published in the open-access journal PLoS Genetics, their findings will make for a fascinating experiment in applied genetics and human nature: what will dog breeders do with this information, and what are the implications for human athletic performance?

A research team led by Elaine Ostrander, chief of the Cancer Genetics Branch in NHGRI’s Division of Intramural Research, reports that a mutation in a gene that codes for a muscle protein known as myostatin can increase muscle mass and enhance racing performance in whippets. Like humans, dogs have two copies of every gene, one inherited from their mother and the other from their father. Dr. Ostrander and colleagues found those whippets with one mutated copy of the myostatin (MTSN) gene and one normal copy to be more muscled than normal and are among the breed’s fastest racers. However, their research also showed that whippets with two mutated copies of the MTSN gene have a gross excess of muscle and are rarely found among competitive racers.

This is the first work to link athletic performance to a mutation in the myostatin gene, with Dr. Ostrander observing: “The potential to increase an athlete’s performance by disrupting MSTN either by natural or perhaps artificial means could change the face of competitive human and canine athletics.” However, the authors stress that: “Extreme caution should be exercised when acting upon these results because we do not know the consequences for overall health associated with myostatin mutations.”

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Article adapted by MD Sports Weblog from original press release.
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Contact: Johanna Dehlinger
Public Library of Science

CITATION: Mosher DS, Quignon P, Bustamante CD, Sutter NB, Mellersh CS, et al. (2007) A Mutation in the Myostatin Gene Increases Muscle Mass and Enhances Racing Performance in Heterozygote Dogs. PLoS Genet. doi:10.1371/journal.pgen.0030079.eor