The method, known as "stable isotope ratio mass spectrometry," can determine where a substance was produced by "weighing" various forms or isotopes of an element in the substance – such as the ratio of rare oxygen-18 to common oxygen-16.
Additional uses of the method may result from a new study that challenges the long-held belief that water moves so rapidly through cell membranes and pores that the water inside cells is chemically identical to the water outside cells.
Scientists from the University of Utah in Salt Lake City and Pacific Northwest National Laboratory in Richland, Wash., published the study the week of Monday Nov. 21 in the online edition of the journal Proceedings of the National Academy of Sciences.
The researchers found that up to 70 percent of the water inside rapidly growing bacterial cells was generated by metabolism, the process of converting food into energy and other necessities of life. That conclusion was based on their surprising discovery that water inside the bacterial cells (intracellular water) has a different oxygen-18-to-oxygen-16 ratio than water outside the cells (extracellular water).
"We've shown a significant portion of the water inside the bacteria can come from metabolism of the food and oxygen they consumed," and not from water outside the cell, says University of Utah chemist Eric Hegg, the study's principal author.
If future research proves the same thing is true in mammalian cells, then the difference in isotopic makeup of water inside and outside of rapidly growing cells might be used to detect fast-growing cancer cells in the brain or other hard-to-biopsy areas of the body, or study the metabolism of obese people or people suffering anorexia or bulimia, says Hegg, an assistant professor chemistry.
Hegg did the study with James Ehleringer, a distinguished professor of biology at the University of Utah, and Helen Kreuzer-Martin, a University of Utah research assistant professor of biology and a staff scientist at Pacific Northwest National Laboratory.
Isotope Ratio Analysis Combats Terrorism
The analysis of stable isotopes – forms of an element that are stable because they do not decay radioactively – started out as a way to learn about how ecosystems work, based on how environmental factors affect the proportions of various isotopes in plants, animals, soil and air. Later, stable isotopes in sediments or other materials were used to learn details about prehistoric environments, such as changing temperatures over time.
In recent years, Ehleringer pioneered the use of stable isotopes to study what has been dubbed "the ecology of terrorism." He has helped the Central Intelligence Agency, Federal Bureau of Investigation, Secret Service and Drug Enforcement Administration.
Several years ago, Ehleringer analyzed counterfeit U.S. $100 bills and showed cotton in the counterfeits had different oxygen isotope ratios than Texas-grown cotton in legitimate bills. Early counterfeits had cotton that isotope analysis indicated was grown in a wet, cool climate, while later versions came from a dry, warm climate. That confirmed the government's belief the counterfeiters had ties with terrorists who moved from Eastern Europe to the Middle East.
In 2000, the Drug Enforcement Administration began using Ehleringer's method to test thousands of drug samples each year. Slight differences in the nitrogen-15-to-nitrogen-14 ratios helped Ehleringer distinguish soils where coca plants were grown, while ratios of carbon-13 to carbon-12 revealed plants grown in humid versus drier climates. That let the DEA distinguish cocaine from Peru, Colombia, Bolivia or Ecuador.
Similar analysis of carbon isotopes in heroin and chemicals used to process it made it possible to learn where heroin poppies were grown and processed.
Ehleringer and Kreuzer-Martin later showed that the oxygen isotope ratios in bacteria similar to anthrax reflected the ratios of the water in which they were cultured, providing clues to where the bacteria were grown.
The method also has been used to track the source of explosives favored by terrorists, and has been proposed as a way to use hair samples from terrorists to determine their past movements, based on isotope ratios in food and water at different locations.
How the New Study Was Performed
Prevailing wisdom says that water inside and outside of cells is identical in terms of ratios of oxygen-18 to oxygen-16, and rare hydrogen-2 to common hydrogen-1.
The paradigm is that water diffuses so quickly into and out of cells that the water inside a cell is indistinguishable from water outside a cell," Hegg says.
The researchers grew E. coli bacteria at body temperature in flasks containing a nutrient-rich liquid culture medium. There were four sets of flasks, each holding a growth medium containing water with a different ratio of oxygen-18 to oxygen-16.
After the bacteria grew for three hours, the contents of each flask were sucked through a filter, leaving a pasty "cake" of bacterial cells with the consistency of wet flour.
Water was extracted from the bacterial cakes by placing each cake in a test tube, freezing the cakes by putting the tubes in liquid nitrogen, using a vacuum to remove the air from each tube, then putting the tubes in boiling water. The water from each cell cake was boiled into steam, which was routed to another tube where the water condensed.
Before undergoing this process, half of the bacterial cakes were washed with water with four different ratios of oxygen-18 to oxygen-16. The washing process allowed researchers to calculate how much of the water extracted from the bacterial cakes came from outside and from inside the bacteria. Water samples were analyzed in a mass spectrometer, which detects the atomic weights of isotopes of oxygen within the water.
The result: The ratio of oxygen-18 to oxygen-16 was different in water from inside and outside the bacteria. The extent of that difference allowed the scientists to determine that 30 percent of the water inside rapidly growing E. coli came from outside the bacteria and 70 percent of the water was produced by metabolism inside the bacteria.
Ehleringer says the new study will not affect most existing uses of stable isotope analysis because the findings apply only to rapidly growing cells like bacteria, and most uses of stable isotope analysis do not involve fast-growing cells.
Hegg and Kreuzer-Martin say the new findings might make it more complicated to determine where bacteria used in bioterrorism were grown. That isn't true of anthrax, which is spread by spores that are almost dormant. But for live bioweapons spread through the air in an aerosol mist – bacteria that cause plague, tularemia or Q fever, for example – the difference in isotope concentrations inside and outside the bacterial cells must be taken into account in trying to identify where the bacteria were grown based on the isotopic signature of the water in which they were cultured.
Findings Suggest a New Way to Study Eating Disorders
"One area where our results could prove very useful is in assessing metabolic activity," Hegg says. "Being able to accurately measure this number is often important in obesity research and understanding eating disorders. The greater the metabolic activity of a cell, the bigger the difference between the 'inside' water and the 'outside' water."
Hegg says a person's metabolic rate now is measured by having them drink water enriched in oxygen-18 and hydrogen-2. Over time, these uncommon isotopes are excreted through urine, and additional oxygen-18 is lost as carbon dioxide is exhaled. The person gradually regains normal levels of the common isotopes oxygen-16 and hydrogen-1, and the change allows the metabolic rate to be calculated.
"As with any scientific measurement, there will be errors associated with this calculated metabolic rate," Hegg says. "Thus, having a second way to measure metabolism would be beneficial."
A potential second method could measure oxygen and perhaps hydrogen isotope ratios in a common metabolite – a product produced by most human cells as they metabolize food and water. The ratios of the isotopes would reveal the percentage of water in body cells that was produced by metabolism, and thus the rate of metabolism.
"We could compare the ratios obtained from a 'healthy' person to the ratios obtained from an obese person," Hegg says. "One also could imagine performing similar tests on an anorexic or bulimic individual. You would be asking whether the isotope ratio of the metabolite more closely matched the food that the individual consumed or the proteins already in the individual's body. If it is the latter, then it means that the individual is basically in a starvation state, living off of their stored proteins."
Finding Hidden but Aggressive Cancers
The difference in isotope ratios in water inside and outside a cancer cell "could be useful in developing a test to assess the metabolic rate of a tumor – how fast the tumor is growing," says Hegg. "This could be especially important in tumors in which obtaining a biopsy is difficult."
If doctors can do a biopsy to remove a sample of a tumor, the cancer cells can be cultured in a dish to determine how rapidly they grow. "But if you can't biopsy it because it's in your brain or another hard-to-reach location, then you need some other way to figure out how fast it's growing," Hegg says.
Such a test would involve taking a blood sample to collect some metabolite produced by the cancer cells.
"If that metabolite has the same isotope ratio as the water in your bloodstream, then the tumor is not growing so rapidly," says Hegg. "However, if the isotope ratio in the metabolite is vastly different than the ratio in your body water, it indicates the tumor is growing very rapidly, so it needs to be treated more aggressively."
Journal
Proceedings of the National Academy of Sciences