By Saren Johnston
For good or for bad -- the jury is still out -- tiny amounts of various metals in our bodies give up their freedom for a chance to cozy up to our DNA. In fact, trace metals can get pretty friendly with all kinds of biological systems, abandoning their freedom in the ecosphere to attach and bind themselves to proteins and other biomolecules. And whether this circumstance is good or bad depends on the element.
"The basic phenomena responsible for the toxicity of trace elements are complex," says Sam Houk, senior chemist. "Some elements like cadmium and lead can replace essential elements like copper and zinc in enzymes, thus interfering with the biological activity of the enzyme.
Radioactive elements like uranium, thorium and other actinides are toxic chemically. They can also bind to DNA and cause mutations when they decay. Since the Department of Energy has large amounts of such elements in storage, monitoring their binding to proteins and DNA at very low concentrations is of great environmental importance."
Using a novel extension of inductively coupled plasma-mass spectrometry, a well established analytical technique pioneered atAmes Laboratory, Houk can determine the total amounts of ultratrace metals in biological specimens. His method is faster and more sensitive and selective than conventional schemes. Allowing measurements at parts-per-trillion levels, the ICP-MS technique relies on a special type of chromatographic separation called size exclusion chromatography in connection with a state-of-the-art magnetic sector mass spectrometer and an inductively coupled plasma -- a very hot argon gas.
In Houk's new method, chromatographic separation of the compounds of interest provides chemical information, in particular the molecular weights of the proteins. Jin Wang, who recently received his Ph.D. with Houk and is now a postdoctoral fellow at Baylor College of Medicine in Houston, did much of the chromatography work. "It's his specialty," says Houk. "He did almost all of the experiments. He's good at both chromatography and ICP-MS."
The chromatography work is crucial because it reveals the percentage of a trace element in a sample that is bound to particular biomolecules. "If we just put the entire sample in the ICP, it would only measure the total amounts of the individual elements present," says Houk. "It would not tell us that, say, 90 percent of an element is in a particular molecular weight range, and 10 percent is in a different fraction."
Once the chromatography is completed, the sample is automatically injected into a nebulizer that produces a mist of fine droplets. The droplets dry into aerosol particles and pass into a hot argon inductively coupled plasma. In the ICP, they are converted to atomic ions that are measured by the mass spectrometer. The resulting measurements provide data that allow scientists to better comprehend the effects of unusual elements in biology and environmental sciences.
Houk is quick to point out that his development of the new ICP-MS method for trace metal identification was facilitated by interactions with instrument manufacturers Transgenomic CETAC Technologies and Finnigan MAT and Micromass Ltd. CETAC provided personnel, nebulizers and supplies, while Finnigan MAT loaned the mass spectrometer on which Houk ran his experiments. And Dan Wiederin, a former Ph.D. student of Houk's and now the president of Elemental Scientific in Omaha, has served as the primary collaborator on the research.
Houk's experiment is applicable to almost any element in practically any kind of biological system. "The main exceptions are the common elements that are always present: carbon, nitrogen, oxygen and hydrogen -- we don't measure those," he says.
Just as there are few limits regarding the elements to which Houk can apply his methodology, there is also a seemingly endless array of potential applications. Work for the Department of Energy deals with determining the fate of radionuclides in the ecosphere, a major concern in the cleanup of nuclear facilities. The conversion of radioactive elements to atomic ions in the ICP considerably reduces the time required for measuring nuclides with long half-lives from that required by conventional techniques.
"There are also important medical applications," says Houk. "For example, there's a lot of interest in selenium compounds, which are being marketed as health foods and as preventive treatments for cancer and are touted to delay the onset of AIDS symptoms. But there have been few direct measurements of the role of selenium in this regard."
Agricultural applications could involve extracting proteins or other macromolecules from grains and measuring the binding of trace elements. Similarly, researchers could measure the binding of metal ions to environmental compounds, such as humic acids, large polymeric compounds with many carboxylate groups attached.
Houk says his experiment could also be used to measure trace metals in synthetic polymers if they could be brought into solution. "There is some interest in this since the processing procedures for polymers usually involve exposing the material to metals," he explains. "There's a question about where the metals wind up in the final product."
Houk's experiment can accommodate just about any organic sample as long as it has an inorganic element in it. But now his goal is to improve selectivity in identifying the biological compounds. "Currently, all we get in this particular experiment is a kind of crude separation of compounds based on their molecular weight," he says. "A chromatographic fraction for an element could include many different proteins that contain that element. We want to separate those proteins more effectively and also develop mass spectrometric measurements that will be sensitive to the particular molecule. You see, we get information on the identity of the molecule only from the chromatography, not from the ICP. What the ICP does is give us an extremely sensitive and selective way to find unique inorganic elements."
To more precisely identify the proteins, Houk says they would probably start with size exclusion chromatography, collect a fraction of the liquid coming out of the chromatographic column and inject it into another kind of chromatographic column that would provide better resolution.
"We might also try capillary electrophoresis, which is an effective way to separate proteins. And we could use different variations of mass spectrometry, such as electrospray MS, which can measure molecular weights and also the sequences of amino acids but is much less sensitive to whether there are small levels of inorganic elements present. These are some of the things we have in mind for the future."
For more information:
Sam Houk, (515) 294-9462
rshouk@iastate.edu
Current research funded by:
DOE Office of Basic Energy Sciences
Last revision: 12/17/99 sd
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