ac detective
Finding acrylamide How Swedish researchers discovered a probable carcinogen in food.
© 2004 AMERICAN CHEMICAL SOCIETY
Hemoglobin + AA (a)
– Val – AA COURTESY OF MARGARETA TÖRNQVIST
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t wasn’t just the dead rainbow trout and paralyzed cows that had residents near Hallandsås ridge in southwestern Sweden concerned in the fall of 1997. About one-fourth of the workers constructing a nearby railway tunnel were experiencing numbness and tingling in their feet and legs. Consumers resisted buying milk, potatoes, and other agricultural products from the area, and the annual hunting season was called off in fear that moose and deer were also contaminated. Scientists were suspicious that a dangerous chemical was leaching out of the 1500 tons of grouting agent used in the Hallandsås tunnel. Closer inspection of surrounding streams and groundwater confirmed that they were right. Analyses revealed high levels of acrylamide, a known neurotoxin and probable carcinogen. Acrylamide was used as a monomer in the grouting agent. Although acrylamide was found in the environment around the Hallandsås tunnel construction site, there was no proof that it was the cause of the dead fish and paralyzed cows, says Margareta Törnqvist of Stockholm University. It just so happened, however, that as a Ph.D. student, Törnqvist developed a hemoglobin (Hb)-adduct method for monitoring reactive compounds like acrylamide in vivo (see figure). A colleague had used the method to measure acrylamide exposure in workers in China, but it wasn’t known whether it would work on cattle and wildlife. “I suggested that we could try this protein-adduct analysis on the animals,” says Törnqvist. Sure enough, the method worked, and without a doubt, it showed that the animals had been exposed to acrylamide. This finding, along with the high content of acrylamide in a nearby stream, led to
(b) AA-adduct (c) Data on dose and uptake
Assessing acrylamide exposure. (a) Acrylamide (AA) forms a stable reaction product or adduct with hemoglobin, which accumulates over the life of red blood cells. (b) Adducts to N-terminal valine (Val) are isolated using a modified Edman degradation and (c) analyzed by either GC/MS/MS or LC/MS/MS. Adduct levels in blood provide information on the average dose and uptake of acrylamide in an individual during the past four months.
discontinuation of the tunnel work. The results also sparked concern about the tunnel workers, says Törnqvist. Using the same method, she and her colleagues examined the exposed workers’ blood and discovered adduct levels as high as 4000 pmol/g Hb. Reference samples taken from people who lived outside the contaminated area contained 30– 40 pmol/g Hb. A closer look at the level of acrylamide in the reference samples revealed another reason for concern. “When we made
risk calculations, we saw that this could be important,” says Törnqvist. The researchers estimated that the background level would correspond to an uptake of ~100 µg acrylamide/adult/day. “If you assume this uptake over a lifetime, it is really high,” says Törnqvist. It became clear that the general population was being exposed to a relatively constant source of acrylamide, but no one knew for sure what it was. What made things even more puzzling was that the background level seen in humans was nearly absent in wildlife from uncontaminated areas, says Törnqvist. The researchers knew that Hb-adduct levels in smokers were higher than in nonsmokers. This suggested that burning or high temperatures might be involved. They began to look at diet and the possibility that heating food was responsible for the elevated background levels of acrylamide in the general population. To test their theory, Törnqvist and colleagues fed rats either fried or unfried feed. The rats that ate the fried feed had adduct levels of 65–70 pmol/g Hb, whereas those that ate unfried feed had very low Hb-adduct levels. The researchers then began to investigate acrylamide in human foods. The discovery of carcinogenic/mutagenic heterocyclic amines in steak in the 1970s by Takashi Sugimura of Japan’s National Cancer Center pointed toward the involvement of proteins in the formation of acrylamide. So, Törnqvist and colleagues first looked at protein-rich foods like hamburger. They found that acrylamide levels increased with temperature, but the levels were not high enough for meat to be the primary source. “You would have to eat two kilograms of hamburger per day,” says Törnqvist.
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ac detective
Next on the list were carbohydraterich foods. Very high levels of acrylamide were found, particularly in potato chips, which had levels up to 4 mg/kg. The results were a little bit shocking, remembers Törnqvist. “We were afraid to publish the results because of the worries associated with the word acrylamide after the Hallandsås incident,” she says. “We really had to present incontestable proof that it was acrylamide and nothing else.” On the other end of the spectrum, boiled and raw foods had no detectable acrylamide. The results suggested that high temperatures (>120 °C) are needed to form acrylamide in foods. The Swedish group’s results were eventually published in 2002, sparking a flurry of media attention and prompting analytical chemists to develop new methods for detecting acrylamide in a wide range of foods. Today, hundreds of foods have been tested for acrylamide. Most analyses are
now performed using LC/MS or LC/ MS/MS, says Törnqvist. Initially, researchers used GC/MS with bromination of the acrylamide, which is a harsher method. “When we used GC/MS, we were afraid that acrylamide or the derivative of acrylamide was formed as an artifact,” she says. LC/MS is milder method and doesn’t involve heat, she adds. The U.S. Food and Drug Administration (www.cfsan.fda.gov), the Swedish National Food Administration (www.slv.se), and a host of other government agencies have now compiled lists of the acrylamide content in commonly consumed foods. The data are freely accessible on the Web. As expected, carbohydraterich foods, including crackers, cookies, potato chips and other snack foods, french fries, breakfast cereals, and even infant teething biscuits, have the highest levels of acrylamide. Surprisingly, prune juice and black olives also contain high amounts. In Sweden, coffee turns out
to be a significant source of acrylamide in the diet, contributing about 30%. “It is not that the level in coffee is particularly high. I think in Sweden we drink rather much coffee and rather strong coffee,” says Törnqvist. Meanwhile, researchers are trying to calculate the cancer risk associated with acrylamide in food. Preliminary estimates suggest that the risk is higher than for any other chemical in food. Currently, the U.S. Centers for Disease Control and Prevention is working to obtain acrylamide exposure data for the general population using an automated, highthroughput LC/MS/MS Hb-adduct method. Experts believe that it is probably not possible to reduce acrylamide in the diet to low levels because of the wide range of foods that are affected. The results, they say, call for new views on cancer risks from natural chemical compounds. a —Britt E. Erickson
books and software Organometallic speciation
Hyphenated Techniques in Speciation Analysis Edited by J. Szpunar and R. L⁄ obin´ski Royal Society of Chemistry, 2004 220 pp, $79.95
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hen it comes to determining the speciation of organometallic compounds by using hyphenated techniques, 248 A
we can all learn a lot from Szpunar and L⁄ obin´ski’s 12 years of research experience. The authors share those experiences in their new book, Hyphenated Techniques in Speciation Analysis. The first section briefly introduces sample preparation approaches, such as derivatization, purge and trap, and solidphase microextraction; separation techniques, such as GC, LC, and CE; and detection methods, such as MS and inductively coupled plasma MS. The introductory chapters are followed by a long list of concise chapters dedicated to several important elements and complex natural matrices. The focus of the chapters reflects the maturity of various research areas. Methods for analyzing methylated species of lead, tin, and mercury are well developed; the dedicated chapters emphasize validated methods that are being implemented for routine determinations.
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The chapters focusing on naturally occurring metallospecies detection emphasize characterization and identification approaches that facilitate exploration of the field. A chapter is also dedicated to the discussion of the importance of quality control and assurance in speciation analysis. The strength of the book is its examples, such as environmental, clinical, natural products, and pharmaceutical areas that involve both liquids and solids, including plant material, animal tissue, and other matrices. The examples also highlight the advantages of the hyphenated techniques for these applications. The book would be a helpful resource for investigators entering the speciation area of research and as a reference for graduate students exploring this topic. Reviewed by Janusz Pawliszyn, University of Waterloo (Canada) © 2004 AMERICAN CHEMICAL SOCIETY
