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Biological Chemistry

Probing For In-Body Ozone

Molecule hunters need better traps to prove that a wily oxygen species is made in the flesh

by Carmen Drahl
June 22, 2009 | A version of this story appeared in Volume 87, Issue 25

Credit: © 2003 Science
Atheronals, with low-density lipoproteins, induce white blood cells to pack themselves with lipids (red), an early step in arterial disaese.
Credit: © 2003 Science
Atheronals, with low-density lipoproteins, induce white blood cells to pack themselves with lipids (red), an early step in arterial disaese.

Paul Wentworth Jr. and his colleagues were studying antibodies when a run-in with oxygen chemistry took them down a completely unexpected path. That diversion led to evidence suggesting that the human body makes ozone, a gas recognized more for its role in the heavens, shielding Earth from ultraviolet light, and in air pollution, than for any role in the body. The body makes several chemically reactive species derived from oxygen, but the idea that ozone could possibly play a part in body chemistry stunned the scientific community.

That was in 2002. The find spurred a flurry of follow-up work and led to renewed interest in how different oxygen species do both good and bad deeds in biology.

"The proposal that ozone is made naturally in the body hit the field like a bombshell. It's a strong oxidant—to have it generated during natural processes was completely unprecedented," says Albert W. Girotti, who studies reactive oxygen species at the Medical College of Wisconsin.

And it could have medical implications. For one thing, in situ generation of ozone in the body would be a new twist for researchers studying reactive oxygen species in inflammatory and degenerative diseases, says Christine C. Winterbourn, who studies reactive oxidants at the University of Otago, in New Zealand. "There's still a lot that isn't known about the role of reactive oxidants in disease mechanisms and how important individual reactions are," she says.

But nearly seven years after the ozone bombshell dropped, whether the human body generates ozone remains uncertain. Researchers have yet to directly observe ozone in biological systems, and it's not clear whether they're pursuing a prizewinning quarry or a molecular Sasquatch.

Despite the new tools available, researchers who spoke with C&EN agree that the question won't be easy to settle.

Wentworth, a chemist at Scripps Research Institute and Oxford University, stumbled onto ozone research while examining the immune system's bacteria-busting activities. In collaboration with Scripps President Richard A. Lerner and several other researchers, Wentworth found that antibodies and white blood cells make ozone and wield it as a weapon in the fight against bacteria (Science 2002, 298, 2195). They later found evidence that ozone contributes to the buildup of fatty plaques in the diseased, hardened arteries of patients with atherosclerosis (C&EN, Nov. 10, 2003, page 12; Science 2003, 302, 1053).

"We use the term 'signature of ozone' in our work," Lerner says. "Unless you can measure ozone directly, that's what you must say," he adds. The Scripps team used three different molecules as diagnostic probes for ozone: 4-vinylbenzoic acid, cholesterol, and indigo carmine. On the benchtop, ozone cleaves a double bond in each probe's structure, leaving behind molecular evidence of its presence. So, experiments that involve trawling the body for these cleaved probe molecules could be telling of ozone's bodily presence.

But follow-up reports from other groups indicate that those lines of evidence aren't completely clear-cut. It's relatively easy for atmospheric chemists to detect ozone in the skies, simply by looking for its spectroscopic signal. But detecting ozone in biology "is like chasing an animal that's extremely fast, with footprints that are almost invisible," says William A. Pryor, a professor emeritus at Louisiana State University who studies reactive oxygen species.

Ozone reacts with olefins in rapid, multistep reactions, Pryor explains. The products from the earliest steps, called ozonides, are excellent indicators of ozone's presence, but they tend to be highly unstable under biological conditions. Subsequent reaction steps lead to more stable products, but unfortunately, they are the same products that one might expect from a reaction with other chemically reactive entities in the body, Pryor says.

That situation has complicated the interpretation of data from each of the Scripps team's three probes. For instance, an enzyme called myeloperoxidase can transform 4-vinylbenzoic acid into 4-formylbenzoic acid, just as ozone would, Winterbourn says. Myeloperoxidase is found in the very white blood cells that the Scripps team studied, so the ozone signature from that probe might be because of the enzyme instead, she says.

As for the molecular probe cholesterol, ozone cleaves its double bond to make an unusual pair of products. The Scripps team dubbed them the atheronals and designated them as molecular indicators of ozone. But atheronals might also come from cholesterol's reaction with singlet oxygen, a high-energy form of molecular oxygen that exists in the body. Independent teams led by Derek A. Pratt of Queen's University, in Ontario, and Paolo Di Mascio at the University of São Paulo, in Brazil, have each suggested a different mechanism for atheronal formation by singlet oxygen (J. Am. Chem. Soc. 2008, 130, 12224; Chem. Res. Toxicol. 2009, 22, 875).

In response to the cholesterol findings, Wentworth's group has suggested a way to differentiate between the ozone and singlet oxygen routes to the atheronals. Product ratios are the key, Wentworth says. His team found that exposing cholesterol to ozone leads to more of one of the atheronals compared with the other. The opposite ratio turns out to be true when cholesterol reacts with other reactive oxygen species common in biology, including singlet oxygen.

With that information, Wentworth's team looked back at their 2003 data from patients' arteries and found that the atheronal ratios in the arteries are suggestive of the ozone mechanism in 20 out of their 28 samples (Chem. Commun. 2009, 3098). However, Wentworth tells C&EN that the team hasn't yet carried out calculations that demonstrate that that difference is statistically significant.

The third probe, indigo carmine, isn't a selective ozone probe either. The Scripps team knew that both ozone and singlet oxygen can convert indigo carmine, which is blue, to a colorless sulfonic acid product. They distinguished those two possibilities by way of a method that relies on isotopically labeled water.

Subsequently, Winterbourn and her colleague Anthony J. Kettle, also at Otago, demonstrated that superoxide, a free-radical species that's commonly found in biology, behaves identically to ozone when indigo carmine is around, including in isotopically labeled water (J. Biol. Chem. 2004, 279, 18521).

That finding might have sunk the possibility of using indigo carmine as a probe for ozone, but Kouhei Yamashita of Kyoto University Hospital, in Japan, and colleagues found a case where it still could be a valid ozone detector. Their findings support and follow up on the Scripps team's work. The Japanese group studied white blood cells incapable of making superoxide. Without superoxide around to complicate the signal from indigo carmine, they were able to detect an entity with the signature of ozone in their biological system (Proc. Natl. Acad. Sci. USA 2008, 105, 16912).

None of this proves or disproves ozone's existence in biology, says Wisconsin's Girotti. "Unless you know how to look for ozone properly and definitively, whether it's produced in vivo is an open issue," Girotti says. "Until somebody can definitively say ozone is generated in vivo, there are going to be skeptics."

Biochemists Giuseppe L. Squadrito and Edward M. Postlethwait, both of the University of Alabama, Birmingham, count themselves among the skeptics. If ozone were formed in living things, it would take an unreasonable amount of energy to do it, they write in an e-mail. What's more, they note, since biological systems are rife with antioxidant molecules that react with ozone far more readily than cholesterol does, the body would need to be making an awful lot of ozone for the atheronals to be showing up in the Scripps team's samples at all.

Figuring out whether the body makes ozone boils down to being able to selectively detect it in biological systems. That's a chemistry problem first and foremost, says Christopher J. Chang, who develops probes for reactive oxygen species at the University of California, Berkeley. "If you can find some chemistry that will allow you to discriminate between similar species, you can adapt it to suit any application or detection scheme you want," he says.

To that end, Wentworth's team is looking for better ozone probes. They'd like to be able to trap the molecular beast as it lays down its earliest footprints. "We're looking for molecules with double bonds that form highly stable ozonides," Wentworth says. "If we can detect stable ozonides in biological systems then the evidence for ozone in biology will be strengthened tremendously."

Other researchers are also making strides in ozone detection. Last month, Kazunori Koide, a chemist at the University of Pittsburgh, reported a fluorescent probe that, in initial tests in cells, glows only in the presence of ozone (Nat. Chem., DOI: 10.1038/nchem.240).

The real amounts of ozone in tissues, if any, might be lower than what was added for this study, and there are plenty of other molecules in cells for any scarce ozone to react with first, Winterbourn says. It'll be exciting to see how well the new probe can compete, she says.

Both the fundamental question of whether the body makes ozone and the possibility that it is a factor in some diseases ensures that researchers will continue their ozone hunt.

"We run on oxygen—as human beings it's how we survive," Chang says. "Any chemistry that's derived from the use of oxygen is intimately tied to our existence."


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