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RESEARCH PROFILES
What if you could determine your cholesterol levels through a simple breath test instead of enduring a needle for a blood test? In an early step toward this goal, Purnendu “Sandy” Dasgupta and colleagues at the University of Texas Arlington, the U.S. Environmental Protection Agency, and Kumamoto University (Japan) report in this issue of Analytical Chemistry (pp 2641–2649) the measurement of isoprene, a compound that’s abundant in breath and plays a critical part in cholesterol’s biosynthetic pathway. Dasgupta got intrigued by breath analysis through his children. “Both of my kids have childhood asthma,” he says. “A new gentleman came into our medical school. One day, when I was chatting with him, he told me [that] by far the best way for early diagnosis of asthma and its management was by measuring breath nitric oxide levels.” That got Dasgupta working on affordable solutions to measure nitric oxide in breath. His focus soon expanded and his group developed a method to quantify ammonia, the only basic gas in breath (Anal. Chem. 2006, 78, 7284 –7291). Isoprene caught their attention because, Dasgupta says, “it’s a very enigmatic compound.” Isoprene makes up as much as 70% of all the volatile hydrocarbons in breath. Although it’s been debated whether unusual levels of isoprene can be linked to diseases, the current body of evidence doesn’t dispute the fact that the level of breath isoprene is related to the endogenous biosynthetic pathway for cholesterol. In fact, the health insurance industry is considering collecting breath isoprene measurements. But current quantitation methods— variations of GC, MS, and GC/MS— require technical expertise and are expensive. So Dasgupta and colleagues took on the Hills–Zimmerman ozonebased chemiluminescence reaction that’s normally used to measure atmospheric 2610
JASON DYKE
Measuring isoprene in breath
Breath is collected in balloons for isoprene measurements.
isoprene and decided to see whether it had the sensitivity to work for breath isoprene. They collected breath in balloons, reacted the gas with freshly generated ozone, and measured the chemiluminescence signal on a photomultiplier tube. They compared the results with those obtained by GC/MS. Ultimately, the reaction worked. Dasgupta says initial attempts to measure breath isoprene resulted in higher readings than those measured by GC/MS. “It was pretty obvious that there were interferences,” he says. “I remember one time we measured a fellow’s breath who loves Dr. Pepper. He came back after a drink of Dr. Pepper, and his breath chemiluminescence signal was sky-high!” A flavoring agent in the soft drink was producing the signal. Dasgupta and colleagues realized that a preconcentration method was needed before the ozone reaction. They transferred breath captured in a balloon onto a warm, carbon-based sorbent column. The warm column didn’t absorb much of the interfering water vapor, ethylene, and propylene in breath, but it absorbed isoprene. The column was
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 , 2 0 0 7
then flash-heated to release the gas for the ozone reaction. “We figured out that the warm column preconcentration gives you the right values for ~50–60% of people,” explains Dasgupta. But “it definitely fails when you have smokers and they have just smoked. There’s still stuff in their lungs that’s active towards ozone.” The investigators decided to go one step further, and just like clinicians who carry out blood glucose tests after the patients fast, they collected breath from their subjects first thing in the morning. “This time we were up to ~80%, but there were always 1 or 2, out of 10 people, whose breath isoprene didn’t match the GC/MS data,” says Dasgupta. “I started looking at the detailed GC/MS analyses to see just what the difference was.” It became apparent that sulfur-based gases in breath, such as H2S, CH3SH, and (CH3)2S, were the culprits. Even if they were present at trace levels, their chemiluminescence signals could be 2 orders of magnitude higher than isoprene’s. So the investigators had to come up with a way to remove them. “Silver nitrate solution is an effective way, but I couldn’t see people making a silver nitrate solution and passing the sample through it,” says Dasgupta. “It had to be something that was a replaceable cartridge. So, we came up with the idea of putting silver on an ion exchange resin cartridge, and it worked.” Now that his group has demonstrated that the cheaper, easier ozone chemiluminescence reaction works for measuring breath isoprene, Dasgupta says, “I really want to set up a collaborative research system with clinicians.” His ultimate goal is to collect breath samples from all patients entering a clinic over a period of a few years. He hopes the detailed breath analyses will eventually establish breath biomarker profiles for certain diseases. a —Rajendrani Mukhopadhyay
