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Researchers measured 200 blood metabolites in samples taken from marathon runners and others.
Image courtesy of ©iStockphoto.com/vndrpttn

The day before the 2006 Boston marathon, cardiologist Greg Lewis and his colleagues were preparing for the big race. Armed with a centrifuge and collection tubes, they took samples from runners to measure the levels of over 200 molecules in their blood. The next day, as these volunteers finished the 26.2-mile race, the researchers were waiting in a tent just beyond the finish line where they collected a second sample for comparison.

Along with samples from two other groups of individuals undergoing short-term exercise testing, the runners’ samples gave the researchers a snapshot of the molecules in the blood before and after exercise and shed light on the way that exercise changes our metabolic makeup. Their results appear in the May 26 issue of Science Translational Medicine.

The researchers tracked entire pathways affected by exercise and found that the level of some molecules increased by up to 1000 percent. They also found that these changes persist long after a person stops exercising. Ultimately, these metabolic differences could help researchers understand how exercise has its salutary effects.

“Emerging metabolic profiling technologies have made it possible to take ‘snapshots’ of a whole organism’s metabolic status. However, no one had previously taken a comprehensive, systematic view of the metabolic response to exercise by simultaneously assaying a large and diverse set of known metabolites,” said Lewis, an associated researcher at the ӳ����ý and instructor in medicine at Harvard Medical School.

Metabolite profiling, or metabolomics, is the study of the levels of the body's naturally occurring small molecules (as opposed to the small molecules that chemical biologists create in a laboratory). These endogenous molecules are called metabolites.

When doctors measure an individual’s glucose levels (an indicator of diabetes) or cholesterol and triglycerides (linked to the occurrence of heart disease), they are engaging in a basic form of metabolite profiling. The metabolite profiling that Lewis and his colleagues conducted was more sweeping. They looked for new connections between metabolites and exercise in hundreds of patient samples.

Clary Clish, director of the Metabolite Profiling Initiative at the ӳ����ý, collaborated with Lewis to measure hundreds of metabolites in the collected samples. “In other experiments, you might just measure glucose or cholesterol. Here, we attempt to measure a number of compounds that are representative of biochemical pathways or biological processes,” said Clish. “This gives us additional windows into what’s happening physiologically in response to exercise.”

Robert Gerszten, director of Clinical and Translational Research at the MGH Heart Center and senior author on the paper, explained that the generation of tools like the ones available at the ӳ����ý has made studies of this scale possible. “It’s only become feasible recently to comprehensively define a biochemical snapshot of what’s going on during exercise with the ultimate goals of identifying things that we can intervene upon and figuring out how exercise confers its beneficial effects,” said Gerszten, who is also a ӳ����ý senior associate member.

Lactic acid build up has been studied before, but the researchers were reassured to see it in their results. Most athletes are intimately familiar with the painful, burning feeling caused when lactic acid accumulates in the muscles in response to strenuous activity. Researchers also were not surprised to see that metabolites reflecting utilization of glucose, carbohydrates, and fat – the body’s fuel sources – were elevated during exercise. “One of the advantages to looking at over 200 metabolites is that you see things you expect to see,” said Lewis. “But with a broad-based approach, you also find unexpected things.”

One of the unexpected findings from the study was the detection of intracellular metabolites – molecules that scientists had previously thought were confined within cells. They found molecules connected to blood vessel constriction and cellular respiration. The researchers also detected increased levels of niacinamide, which helps control insulin levels in the body.

The researchers observed that these changes persist long after exercise is over. In addition to collecting samples from marathon runners, the researchers profiled individuals referred for exercise testing at Massachusetts General Hospital, where the participants exercised on a treadmill or bicycle. In addition to taking measurements before exercise and at the peak of a workout, the researchers measured metabolites an hour after subjects ran or biked.

“An hour after they’ve completed ten minutes of exercise, (the subjects’) blood pressure and heart rate have returned to normal and they’re back to going about their daily activities, yet the metabolic changes that we see at peak exercise largely persist when we measure metabolites again,” Lewis said.

Obtaining samples from marathon runners was more challenging for the team since it required drawing blood the day before and the day of the race. But Lewis, who ran the marathon in 2002, says the samples allowed them to see unique patterns of metabolite changes in more and less fit individuals. “It took a lot of effort by a dedicated team to get those samples, but they were well worth it in terms of the metabolic changes we see,” he said. “For the marathon, the metabolic changes are profound.”

In addition to taking a snapshot of the molecules influenced by exercise, the researchers wanted to look at these metabolites’ effects. To do so, Lewis and Gerszten designed experiments with colleagues from Beth Israel Medical Center (Zolt Arany and Glenn Rowe) to sprinkle metabolites onto cultured muscle cells. A cocktail of metabolites that were altered by exercise had an effect that no single molecule could produce: the combination of metabolites induced a transcription factor called nur77, which controls how glucose and lipids are used in the body.

“That experiment raises some interesting questions: how important is that transcription factor to meeting the energy demands of exercise?” said Clish.

The inspiration for studying the influence of exercise on metabolites came more than ten years ago. In addition to being a 2002 Boston marathon participant, Lewis is a rower and was a member of the 1996 US Olympic team. He took a year off from medical school to train for the Olympics, and when he returned to school, one of his scientific mentors invited him to give a lecture on exercise physiology. “That served as the impetus for me to study exercise physiology, which I continue to do today, and it was certainly an impetus to look at the metabolic profile of exercise,” he said. “This has been a long-standing interest of mine.”

Lewis and his colleagues are interested in building on the paper’s findings. If they can determine an optimal metabolic “signature” – a pattern of increased and decreased metabolites – that indicates health and fitness, researchers could test the effectiveness of different kinds of diet and exercise regimens. “We’re most interested in helping people who have cardiovascular or metabolic diseases,” said Gerszten. “One of the key steps downstream will be to identify which of these metabolites are rate-limiting or depleted during exercise” with the goal of replenishing them.

Researchers are also interested in looking at how metabolites change in response to people consuming calories instead of burning them. Gerszten and his colleagues have already begun collecting data. “Ultimately, we want to start integrating these data with genetic data as well,” said Gerszten.

For the current study, the researchers used a technology called liquid-chromatography tandem mass spectrometry (mass spec for short). Mass spec technology is flexible, allowing scientists to measure a variety of molecules like sugars, amino acids, and central metabolites and is also sensitive enough to detect subtle changes. Clish credits the group’s ability to undertake this experiment to the combination of mass spec technology and MGH’s unique facility where Lewis was able to take a variety of physical measurements. “If you marry that technology to this very nice opportunity for access to well-phenotyped patients, it makes for a great project and obviously some interesting results came from it,” he said.

Lewis was likewise grateful to be able to collaborate with Clish’s team. “The great thing about working along side outstanding scientists at the ӳ����ý is that it enables us to bring a lot of tools to bare,” he said. “I spent my post-doc at the ӳ����ý, under the mentorship of Robert Gerszten, helping establish the metabolite profiling platform with an eye toward applying it to exercise in humans. This is really the culmination of several years worth of work with the ӳ����ý and a multidisciplinary team that made it possible to study all of the cohorts we recruited.”