Detecting Irradiated
FOODS: Use of Hydroxyl Radical &O~BIOMARKERS Lisa R. Karam and Michael G. Simic
National Institute of Standards and Technology Ionizing Radiation Division Building 245/C214 Gaithersburg, MD 20899
Recent legislation in the United States has increased the probability of using ionizing radiation for preserving food. The possible increased use of food irradiation in this country, in addition to current use of the technique in other countries, makes it important to develop a method whereby the extent of irradiation of foods can he determined. Both opponents and proponents of this particular food-processing technique support postirradiation dosimetry (PID) as a way to measure the extent of changes in irradiated products. Toprevent tamperingand alteration of the dosimeters, the best postirradiation dosimeters are those that are inherent in the product exposed to the ionizing radiation. Therefore detection of the intermediates and subsequent products arising from the interaction of ionizing radiation with hiomolecules in foods should be a viable means by which the irradiated status of a food sample can he determined. T o be useful as hiomarkers, however, the p r d ucts formed by irradiation must he detectable hy routine analytical methods, This article not subjen lo US. CopyrigM Published 1988 American Chemical Society
formed exclusively by ionizing radiation (unless formation from alternate methods can he readily determined), and stable for the duration of the expected shelf life of the food product. Method development At the National Institute of Standards and Technology (formerly the National Bureau of Standards), we are developing ways to identify the irradiated status of meats and shellfish. These procedures, based on the detection of certain radiolytic intermediates (e.g., free radicals in hone and hard tissue, detected by electron spin resonance) and products (in protein, detected by mass spectrometry), require sensitive analytical techniques because such substances exist in trace amounts. Using boneless chicken as a model for PID in meat, we solved several challenging analytical problems. An advantage of using irradiation to preserve foods is that, a t the dose levels used, the sample is essentially indistinguishable from its nonirradiated counterpart. This is particularly true of fresh meats, which, a t an exposure dose of 50.1 Mrad (1 kGy) appear, smell, and taste as if they had not been irradiated. Because the irradiated product is so similar to its nonirradiated counterpart, however, distinguishing the two is not easy. More than 50% of the weight of raw meat is water. Therefore, when meat is
exposed to ionizing radiation, the nonaqueous components (principally proteins) undergo changes attributable to “indirect effects” of radiation. That is, the incoming radiation first interacts with molecular water, splitting it into hydroxyl radicals (.OH) and hydrated electrons (eJ. HIO - - O H +
e“;
t H’
In turn, these reactive species interact with the amino acids found in the proteins. The resulting chemical alterations of the amino acids, particularly of p-tyrosine and its nonhydroxylated precursor phenylalanine, have been extensively studied in model systems. Hydroxyl radicals formed by the radiolysis of water interact with the aromatic rings to afford hydroxylated products. Because these products are unique to the irradiated systems (i.e., the products are present in subcritical concentrations in nonirradiated meat), they can act as internal dosimeters, the amount present reflecting the amount (“dose”) of radiation absorbed. Hydroxylation of phenylalanine leads to the production of (2,3-,and 4hydroxypheny1)alanine (0-tyrosine, 1; m-tyrosine, 2; and p-tyrosine, 3; respectively). Likewise, the hydroxylation of p-tyrosine [(4-hydroxyphenyl)alanine] leads to the formation of (3,4-dihydroxyphenyl)alanine (dopa).
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Because dopa is found in the synapses of nonirradiated tissue, it is not a useful product to monitor. Likewise, p-tyrosine is incorporated into proteins and is inappropriate as an internal dosimeter. The 2- and 3-hydroxylated isomers of phenylalanine, however, are suitable as markers of irradiation; they are not routinely incorporated into the protein structure because of the high specificity of tRNA. Since (3-hydroxypheny1)alanine is difficult to separate chromatographically from (4-hydroxypbenyl)alanine, (2-hydroxyphenyl)alanine was chosen as the internal dosimeter.
X = OH 1,Z-OH 2, 3-OH
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Analysis Cubes of fresh chicken breast (-50 g each) were irradiated in a W o - y source (dose rate: 19.85 kradmin; dose range: 0.05-8.0 Mrad) in the presence of air and at ambient temperature, then dried and pulverized with a mortar and pestle. Approximately 5-10 mg of each sample was hydrolyzed with acid. The intemal standard, (4-hydroxyphenyl)glycine (1.00 X mol), was added to each sample before hydrolysis. The hydrolyzed samples were dried and treated with bis(trimethylsily1)trifluoroacetamide (BSTFA) to convert the amino acids to their more volatile trimethylsilyl derivatives. Separation on a fused-silica capillary column using selective ion monitoring (SIM) with a mass-selectivedetector interfaced to a microprocessor-controlled gas chromatograph increased the selectivity and sensitivity by focusing the mass spectrometer on specific ions. The major difficulty in studying biological systems such as chicken meat lies in the complexity of the mixtures. Because we wished to quantify and identify miniscule (parts-per-million) amounts of a specific amino acid, selective ion monitoring was ideal; it can be used to monitor only those ions formed from the fragmentation of the compounds of interest [in this case, those of (2-hydroxypheny1)alanine and of the (4-hydroxypheny1)glycine standard]. Although many trimethylsilyl derivatives of biologically derived molecules share similar fragmentation ions with either (2-hydroxypheny1)alanine or (4-hydroxyphenyl)glycine,the retention times are different. Therefore the compounds of interest can be differentiated chromatographically. Examples of SIM chromatograms of characteristic (2-hydroxypheny1)alanineions appear in Figure 1. By using authentic materials, we de1118A
Figure 1. Gas chromatographyhnass spectrometry-selective ion monitoring chrcmatcgram of (2-hydroxyphenyl)alanine (eTyr, 1) in control and irradiated chicken breast after HCI hydrolysis and trimethylsilylation. (2-hydmxypheoylhlanIne inns: (M
- 15)t m/r 382.2: (M - 117)t rmz 280.2; and (M - 43)t mlr 354.2.
Figure 2. Yield of (2-hydroxyphenyi)alaine (eTyr, 1) in irradiated chicken bret after irradiation, HCI hydrolysis, and trlmethylsilylation. Values are also shown tw me c o n ~ l l m of (2nydmxyphenvlhlanine ( ~ T f l .1) in da alglnal. m d r M chldlen sample (based on Uw arsumpilon hat Ehlcken mal 16 appoxlmately 50% water and 30% fat and oMer nonhydmlyzedcwnponents).
ANALYTICAL CHEMISTRY. VOL. 60, NO. 19, OCTOBER 1, 1988
termined the retention times and fragmentation patterns of the trimethylsilyl derivatives of both (2-hydroxypheny1)alanine and (4-hydroxyphenyl)glycine. The characteristic ions of the fragmentation of the trimethylsilyl derivatives of (2-hydroxypheny1)alanine (M?. mlz 397;(M - 15)?, mlz 382; (M - 43)?, mlz 354; (M 117)?, mlz 280) and of (4-hydroxypheny1)glycine (M?, mlz 383; (M -15)?, mlz 368; (M 43)t. mlz 340; (M 17V. isotope peak, mlz 267) were monitored hy SIM in the 1-min window of the retention times of both (2-hydroxyphenyl)-alanineand (4-hydroxypheny1)glycine (oven temperature 180-190 “C). The amount of (2-hydroxypheny1)alanine in irradiated samples was determined bycomparing the integrated areas of its fragmentation ion peaks with the corresponding peaks of (4-hydroxypheny1)glycine. Yields of (2-hydroxypheny1)alaninein each sample were calculated by using the equation: Y = k A(o-Tyr). mol(0HPC) A(0HPG). mg where Y is the moles of (2-hydroxyphenybalanine formed per milligram of irradiated chicken (after drying), k is the relative molar response factor of each (2-hydroxypheny1)alanine fragmentation ion with respect to its corresponding (4-hydroxypheny1)glycine ion, A(o-Tyr) is the integrated peak area of a given (2-hydroxypheny1)alanine ion, A(0HPG) is the area of the corresponding (4-hydroxypheny1)glycine ion, mol(0HPG) is the number of moles of (4-hydroxypheny1)glycine added (1.00 X and mg is the weight, in milligrams, of dried chicken. Figure 2 illustrates the dose-yield plot for irradiation doses of 0 (control), 0.5,1.0,2.5,4.0, and 5.0 kGy. To obtain the yield of (2-hydroxypheny1)alanine in mglmg of chicken, the number of moles of (2-hydroxypheny1)alanine is multiplied by 181 X lo3 m g h o l e (the molecular weight of tyrosine). During preliminary studies we attempted to remove the lipids by extraction with carbon tetrachloride after irradiation but before drying. This extraction provided a “cleaner” (and therefore less viscous) sample; however, the use of carbon tetrachloride and of certain other organic solvents seemed to induce the formation of (2-hydroxypheny1)alanine in meat samples via a Haber-Weiss-Fentontype generation of .OH:
-
-
-
.
Mitochondria
5-0,
.1 H,O, R(III, .OH Such nonradiolytically generated hydroxyl radicals react with phenylala-
nine in the same manner as those produced by ionizing radiation. Once we identified the background levels of (2-hydroxypheny1)alanine (=12 ppm in nonirradiated chicken meat), the carbon tetrachloride extraction step was eliminated. Because of the higher levels of lipid material in such nonextracted chicken samples, greater care had to he taken during the hydrolysis procedure to reduce foaming (i.e., samples were more finely ground and were cooled in liquid nitrogen to approximately 0 “C during the hydrolysis). In addition, the packed glass sleeve of the gas chromatograph1 mass spectrometer injection port (which acts as a sample filter before the capillary column) had to be repacked more frequently. With these modifications, background levels of (2-hydroxyphenyl) alanine were below the level of detection (50.1 ppm) in nonirradiated samples and the dose-yield of production in irradiated systems was as found in Figure 2. This experiment was performed on three different chickens from December 1985 to October 1986, and in each case, yields were comparable at each dose point. Each sample was analyzed
four times (four injections) by SIM for each ion. The yields at each data point were determined as the average values of each run for all ions in each chicken sample. Levels of (2-hydroxypheny1)alanine may he useful as dosimeters in other meats and foods containing proteins. Additionally, (2-hydroxypheny1)alanine may be used as a biomarker for :OH radical generation in vivo in the absence of any radiation. This latter application may prove to be considerably more important than its contribution to postirradiation dosimetry.
Dizdaroglu, M.; Simic. M. G . In Oxygen Radicals; Rodgers, M.A.J.; Powen, E. L.. Eds.; Academic Press: New York, 1981; pp. 619-21.
Karam. L.R.: Simic, M. G. “Health Impact, Identification. and Dosimetry of Irradiated Foods. Report of a WHO Working Croup on Health Impact and Control Methods of Irradiated Foods”; World Health Organization: Neuherherg, F.R.C., 1988. Karam, L. R.; Simic, M. G. submitted for publication to Riochem. Rioph~ys. . . Res. Cammun.
McCord, J. M.; Fridovich, 1. J. B i d . Chem. 1969,244,6049-55.
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Lisa R. Karam received a E.S. degree (1982) in biology and biochemistry from Berry College, GA, and M.S. (1983)ond Ph.D. (1985) degrees in chemistry from American Uniuersity, Washington, DC. She is a staff scientist at the National Institute of Standards and Technology (NIST) as well as a guest scientist at the Imperial Cancer Research Fund (London). Her research interests include the study of biochemistry of Ataxia Telangiectasia, the manipulation and utilization of plasmids, the effects of ionizing radiation on DNA and proteins, and the adaptation of conventional chromatography and mass spectrometry to the study of biological systems. Michael C.Simic received a Ph.D. degree (1964) in radiation chemistry from Durhom University. England, ond a D.Sci. degree (1984) in free radical processes from the Uniuersity of Newcastle-on-Tyne, England. He moued to the United States in 1968 and worked on the mechanisms of radiation biology a t the Uniuersity of Texas in Austin and on the chemistry and technology of irradiated foods at the U S . Army Natick Research and Development Labs. Since 1980 his work a t the NIST has focused on developing a program dealing with the molecular mechanisms of biological effects of radiation and deueloping specific biomarkers of radiation and free radicals. ANALYTICAL CKMISTRY. VOL. 80. NO. 19. OCTOBER 1. 1988
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