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First biomarker for human sleepiness identified in fruit flies

Scientists have identified the first biochemical marker linked to sleep loss, an enzyme in saliva known as amylase, which increases in activity when sleep deprivation is prolonged.

Researchers hope to make amylase the first of a panel of biomarkers that will aid diagnosis and treatment of sleep disorders and may one day help assess the risk of falling asleep at the wheel of a car or in other dangerous contexts.

"As we prepare for the holiday season and long drives to distant relative's houses, I hope this finding will get people thinking about the dangers and costs of sleep deprivation," says lead author Paul J. Shaw, Ph.D., assistant professor of neurobiology. "If you're feeling sleepy on your way over the river and through the woods to grandmother's house, it's much better to pull over and find a place where you can sleep for a while than to continue on and risk a serious accident."

The study appears this week in the online edition of Proceedings of the National Academy of Sciences. Shaw's lab was the first to show that fruit flies enter a state of inactivity comparable to sleep. They demonstrated that the flies have periods of inactivity where greater stimulation is required to rouse them. Like humans, flies deprived of sleep one day will try to make up for the lost time by sleeping more the next day, a phenomenon referred to as increased sleep drive or sleep debt.

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To identify a marker for sleep debt, Shaw decided to look in saliva. Easily accessible, saliva contains many of the substances found in blood and urine, making it an increasingly popular target for diagnostics. Saliva was also an attractive target for Shaw's lab because the brain areas that regulate sleep drive are known to send signals to the brain areas that regulate salivation.

To start his search, Shaw subjected the flies to different kinds of sleep deprivation and used microarrays to look for changes in activity in many different genes. Amylase levels consistently changed after sleep loss. Amylases are a family of enzymes found in the saliva that break down starch.

To verify amylase's connection to sleep loss, Shaw's lab monitored its activity level after sleep deprivation in different fruit fly lines genetically altered to modify their sleep drive.

In one key test, amylase did not increase in a fly modified to endure sleep deprivation longer than normal flies without incurring sleep debt. When scientists kept the same mutant flies awake for extended nine or 12 hour stretches that normally cause them to incur sleep debt, their amylase levels increased.

"This helped prove that the increases in amylase activity level we were seeing weren't just triggered by wakefulness," Shaw says.

Humans kept awake for 28 hours also had increased amylase levels versus controls allowed to sleep normally.

Shaw's lab previously showed that they can use caffeine and methamphetamine to keep flies awake. Caffeine inflicts sleep debt, causing flies to sleep for extended periods when it wears off, while methamphetamine does not. When they monitored fly amylase levels in response to these drugs, they found caffeine drove amylase activity up while methamphetamine did not.

Flies dosed with the herbicide paraquat did not have increased amylase levels, suggesting changes in amylase activity were not related to stress. Flies lacking the gene for amylase had normal sleep and waking cycles, showing that while amylase is tightly linked to sleep drive, it is not actively involved in its regulation.

"We're very pleased with how tightly amylase levels correlate with sleep debt, but for a good diagnostic test we're likely going to need more than one biomarker," Shaw says. "So we're going to continue to use the processes that we've developed to look for other substances that change in connection with the level of sleep debt."

Stephen L. Duntley, M.D., associate professor of neurology and director of the Washington University Sleep Medicine Center, is a frequent research collaborator with Shaw.

"Despite the tremendous medical and public health consequences of sleep debt, its measurement in humans relies upon unreliable subjective rating scales and expensive, often impractical sleep laboratory testing," Duntley says. "Simple, easily accessible biomarkers for sleep debt in humans would revolutionize our ability to conduct research on the causes and consequences of sleep deprivation and provide clinicians with valuable new tools for diagnosing and assessing treatment efficacy in patients with sleep disorders."

According to Shaw, sleepiness biomarkers will also prove useful to studies of sleep in animals.

"Cetaceans like killer whales, for example, are known to go for extended periods of time without sleep, and we'd like to know more about how that works and whether they incur sleep debt," Shaw says. "Until now, the main way to study sleep deprivation's effects on the brain has been to attach electrodes, which can be a bit awkward when your target is a killer whale. Hopefully the markers we develop will make these kinds of phenomena much easier to study."

Michael C. Purdy | EurekAlert!
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