Malonaldehyde

Content oÃ-Food1 RAYMOND J. SHAMBERGER, BARBARA A. SHAMBERGER ANDCHARLES E. WILLIS Department of Biochemistry, The Cleveland Clinic Foundation, and The Cleveland Clinic Educational Foundation, Cleveland, Ohio 44106

ABSTRACT Several types of commercially available food both cooked and uncooked were tested for the presence of the carcinogenic initiator and mutagen, malonaldehyde, which is a breakdown product of unsaturated fatty acids. The thiobarbituric acid derivative of malonaldehyde from meat extract was identified either by thin layer chromatography or by gas chromatography after silylation. Malonaldehyde was also identified directly by gas chromatography. Among the meats purchased from super markets, beef had the greatest amounts of malonaldehyde. Turkey and cooked chicken had high levels. Most cheeses had only small amounts of malonaldehyde. In contrast, many vegetables and fruits had either minute amounts or no malonaldehyde. J. Nutr. 107: 1404-1409, 1977. carcinogenic initiator INDEXING KEY WORDS malonaldehyde •peroxidized food •mutagen •beef Malonaldehyde, a decomposition prod uct of peroxidized polyunsaturated fatty acids, has recently been shown to be a carcinogenic initiator on mouse skin ( 1 ). Malonaldehyde is also mutagenic (2). Consumption of peroxidized food by ani mals has been related to experimental heart disease, cancer and aging (3-7). The test for malonaldehyde has been used for years to measure the wholesomeness and freshness of foods. High levels of malonaldehyde are generally found in rancid foods (8). Malonaldehyde has been detected in fish meat (9), fish oil (8, 10), rancid salmon oil (11), rancid nuts (12), orange juice essence (13), vegetable oils (14), fats (15), fresh frozen green beans (16), milk (17), milk fat (18), rye bread (19), raw ground beef (20), cured meat (21) and cooked meats (22). The malonaldehyde content of non-rancid food or cooked food has not been determined. Identification of malonaldehyde has for the most part been made by complexing malonaldehyde with thiobarbituric acid in acid solution and then measuring the re sulting red color at 535 nm. Because

colorimetrie measurements can be nonspe cific, malonaldehyde identification could be questioned on this basis. Therefore, other chemical ways to identify malonaldedehyde would be desirable. Because the test for malondehyde has been used mainly to test for rancid food, the purpose of this study was to deter mine whether malonaldehyde is also pres ent in various raw and cooked meats and other foods purchased from supermarkets. A further purpose was to identify chemi cally malonaldehyde. EXPERIMENTAL

Malonaldehyde was determined by the modified distillation method of Tarladgis (18). Fifty milliliters of distilled water and 50 ml of homogenate were poured into a 250 ml round bottom flask. The ho mogenate was prepared by adding 5 g of sample ( chosen from lean areas in the case

Received for publication September 7, 1976. 1This article Is the fllth of a series on antioxldants and cancer. Reprint requests should be addressed to Raymond J. Shamberger, Dept. of Biochemistry, The Cleveland Clinic. 9500 Euclid Ave., Cleveland, Ohio 44106. 1404

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MALONALDEHYDE IN FOOD

of meat) to 50 ml 0.97«saline and then homogenizing the material for at least 3 minutes with a homogeni/er.- The round bottom flask was attached to a Claisen distilling head, which was connected to a water-cooled condenser. After the mixture of water and homogenate was acidified with 5 ml of concentrated HC1, the round bottom flask was heated with a Bunsen burner and 50 ml of distillate was col lected. Thiobarbituric acid reductants were de termined directly on the distillate. Four milliliters of the distillate was added to 4 ml of 10% trichloracetic acid (TCA) in a test tube. One milliliter of 17 w/v thiobarbituric acid (TEA) was added to 4 ml of this solution and this mixture was boiled for 15 minutes. The resulting red color was measured at 535 nm, and also at 515 nm and 555 nm, so that an Allen correction could be made for nonspecific absorption by other tissue components. Blanks which included 2 ml distilled water, 2 ml of 107 TCA, and 1 ml TEA solution were also boiled and measured in the same way as the sample. Blank values were subtracted from the sample readings. Precision on duplicate samples was good. Variation on duplicates ranged from 0 to 4% for 10 meat samples whose values were between 5 and 20 /¿g/g. Twenty replicate analyses from the same piece of beef had a mean ±so of 10.7 ±0.44 ng/g. The coefficient of variation was 4.1%. Standards were prepared from malonaldehyde bis-( dimethylacetal ) .:i Malonaldehyde is released when the diacetal is boiled in acid. Recovery of malonaldehyde added either to water blanks or meat ex tract ranged from 807 to 85 c/( in our modified method as compared with 667 to 707> previously reported by Tarladgis (18). The 107> meat extract in saline was also analyzed directly without distillation. The extract was mixed with an equal amount of 107 TCA and filtered through What man No. 1 filter paper. Four milliliters of filtrate were added to 1 ml of 17> w/v thiobarbituric acid and processed as de scribed above. No difference in quantitation could be found between this gentler method for meat and the distillation method. Similar results have been ob

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tained by Vyncke (23). Vegetables could not be directly done this way because of colorimetrie interference. Three homogenizations were also done under nitrogen. No quantitative differences were observed when compared to homogenizations in air. The structure through which malonalde hyde was bound in food is not known. By adding acid even with the less vigorous conditions outlined above, perhaps malon aldehyde was created as an artifact of the isolation procedure. However, after a meal, partially digested food is presented to the stomach which secretes hydrochloric acid at pH 1. Therefore, physiological con ditions at pH l should parallel our more gentle acid isolation procedure and malon aldehyde should be released in the stomach. Standard TEA derivatives of malonalde hyde and beef extract were chromatographed on Silica Gel G in butanol :acetic acid:water 4:1:5. An identical Rf of 0.81 was observed for both the beef and stan dard after 4 hours. Only one spot was observed. Food samples from food purchased in supermarkets were taken just before cook ing and also when cans were opened. In some cases, samples of meat were collected after cooking and after storage in a refrig erator for 24 hours. At least three different meat samples were taken for each experi mental condition. Variation between these three samples was large in some cases and small in other cases. Because of the small number in each group, the values were averaged. There was less than a 107o dif ference in the means from two cuts of beef from each of which four samples were taken from different areas of lean meat. Fat contained little or no malonalde hyde. A total of about 70 meat samples were included in this study. Other samples such as peanut butter, were taken from a freshly opened jar and from a jar which had been opened several times. Malonal dehyde was determined on two different samples of each fruit and vegetable. The TBA derivative of malonaldehyde was isolated from ground beef by washing with chloroform the aqueous red complex 2Tekmar Model SOT with a 100 Enshaft. * Aldrlch Chemical Company, Milwaukee, sin.

Wiscon

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SHAMBERGER

TABLE 1 Comparisons of the colorimetrie and the gas chromagraphic measurements of maUmaldehyde from beef extract MalonaldehydeColorimetrieMS1.0 Chromatographiert?1.1

14.0 27.5Gas

13.5 26.9

formed by boiling the TCA extract of beef with TEA. The TEA complex was not soluble in chloroform. The TEA complex was extracted with butanol and then dried with nitrogen. One microgram of malonaldehyde measured colorimetrically of both the unknown from beef extract and the standard TBA-malonaldehyde derivative were silylated,4 dissolved in chloroform, and applied to a gas Chromatographie column. Silylating makes the TEA com plex soluble in chloroform. Both chromatograms were recorded5 and showed one major component and two minor com ponents with the same retention times. The gas Chromatograph " used was equipped with a flame ionization detector. The columns were 2 meters by 1.27 cm (2 mm internal diameter) glass containing 10% OV-101 on H.P. Chromosorb W (AW-DMCS).7 The gas Chromatograph was used with the following operating conditions: injection port temperature, 290°;column temperature, 140°;flow rates ml/minute hydrogen, 20: nitrogen, 20; and air, 240. Malonaldehyde was identified directly using gas Chromatograph 8 with a 2 meter by 3.2 mm steel column packed with Pora Pack Q + R (50%, 50%).»The gas chromatograph was used with the following operating conditions: injection port tem perature, 275°; detection temperature, 230°;column temperature, 140°;flow rates ml/minute hydrogen, 20; nitrogen, 20; and air, 240. Peak heights were measured and calculated by computer. Malonaldehyde was also measured on the same samples using the TEA colorimetrie reaction. Peak

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AND WILLIS

heights of standard malonaldehyde in three samples of beef extract matched in direction and quantity with the colori metrie test (table 1). Similar results were found in a second experiment. RESULTS Several cuts of beef contained various amounts of malonaldehyde (table 1). After cooking beef, malonaldehyde levels increased in some cuts and decreased in others. Malonaldehyde levels also in creased when the cooked beef was refrig erated 1 to 2 days. Several uncooked cuts of pork had rela tively low levels of malonaldehyde (table 2). In one experiment, malonaldehyde levels decreased when pork chops were broiled. In two instances when the pork chops had a crumb covering, significant increases of malonaldehyde were observed after broiling. Because the fat of pork is more unsaturated than that of beef, more malonaldehyde should be formed in pork than beef. Instead much less malonalde hyde was found in pork than in beef. This difference could be explained by local processing or by differences in handling pork and beef. Malonaldehyde levels were high in two samples of turkey, but rela tively low in uncooked chicken. Large in creases of malonaldehyde were observed after cooking the chicken. Fresh, fresh-frozen or canned vegetables contained either no malonaldehyde or very low amounts in two separate analyses. Vegetables tested included asparagus, brown beans, beets, broccoli, carrots, cauli flower, celery, green beans, green pepper, Italian green beans, lettuce, mushrooms, onions, peas, potatoes, spinach, squash, tomatoes, yams, and zucchini. Fresh or canned fruit also contained little or no malonaldehyde. The fruits tested included apples, apple sauce, bananas, cherries, grapefruit, oranges, orange juice, pears, pineapple, plums, strawberries, and water melon. The amounts of malonaldehyde in two different samples of dried raisins were «REGISIL (BSTFA + 1% TMCS) Bistrimethyl (silyl) trlfluoroaeetamide, Regis Chemical Company. 5 Varian dual pen recorder, Model 4020. »VarÃ-an2100. 'Applied Science Laboratories, Inc., P.O. Box 440, State College. Pennsylvania. 8 Hewlett Packard. "Alltech Association, Arlington Heights, Illinois.

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MALONALDEHYDE IN FOOD TABLE 2 Malonaldehyde levels in different types of meat1

Cut of meat

Treatment

Sirloin steak1

Uncooked Broiled at 450°for 25 minutes Fat only

Ground sirloin

Uncooked Broiled at 450°until medium rare Cooked and refrigerated 1 day

2.7±0.1 3.9±0.2 5.6±0.4

Round steak1

Uncooked Cooked at 325°for 1.5 hours Cooked and refrigerated 1 day

7.2±1.0 3.7±0.2 5.4±0.2

Ground beef1

Uncooked

6.5±0.4

Rolled rump roast1

Uncooked Cooked 4 hours in water 250° Cooked and refrigerated 2 days

1.4±0.2 0.3±0.1 0.8±0.1

Round steak1

Uncooked Cooked, braised2

1.2±0.4 5.8±2.1

Veal1

Uncooked Cooked, braised

13.9±4.0 1.3±0.2

Sirloin tip roast1

Uncooked Cooked 2 hoursat325°

9.4±3.1 27.0±6.3

Ground round steak1

Uncooked Broiled at 450°for 15 minutes

3.8±0.4 10.4±2.1

Pork chop1

Uncooked Cooked at 425°for 1 hour with crumb covering

1.3±0.2 8.1±2.3

Hot dog (beef1 and pork mixture)

Uncooked Cooked

0.5±0.1 0.5±0.1

Pork chops1

Uncooked Broiled at 425°for 1 hour

1.2±0.2 0.4±0.1

Pork chops1

Uncooked Broiled at 425°for 1 hour with a crumb covering

4.1±0.3 11.1±2.7

Turkey

Uncooked Cooked at325° for5 hours

10.8 9.1

Turkey

Cooked at 350°for 3 hours and refrigerated 1 week

13.5

Cooked at350° for1.5hours

Uncooked

7.7±2.1 39.0 ±8.0

Uncooked Cooked at350° for1 hour

4.0±0.8 20.6±5.2

Chicken1 Chicken1

1Mean ±SEof three separate experiments. simmered for 2 hours.

2 Fried in corn oil for 5 minutes,

1.4 and 2.1 /tg/g. Several miscellaneous foods were also tested. Those with 1.0 /¿g/g of malonaldehyde or more were tomato ketchup (1.0), two samples of

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13.7±2.7 11.0±2.0 0.4±0.1

1 cup of water added ;

French dressing (1.0 and 1.4), and two samples of chopped walnuts (2.0 and 6.4). A freshly opened jar of peanut butter had no malonaldehyde, but after the jar had

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SHAMBERGER, SHAMBERGER AND WILLIS

been opened and in use for some time, 1.2 /*g/g malonaldehyde was found. Vegetable oil and corn oil margarine had no detecta ble malonaldehyde. Natural antioxidants in these oils such as vitamin E may pre vent breakdown of the high levels of unsaturated fatty acids to malonaldehyde. Cheeses had the following amounts of malonaldehyde: American diet cheese 5.7; American cheese 3.6; Swiss cheese 0.3; mozzarella 0.5; muenster 0.0; and ricotta 0.4. Two samples of milk, sour cream, half and half were 0.0. Seafood contained gen erally small amounts of malonaldehyde: lox 0.6; fried sole 1.1; baked lobster 1.7; baked white fish 4.7 and two samples of fried perch 1.2 and 2.6 /¿g/g. DISCUSSION Measurement of malonaldehyde by use of a TEA derivative has been used for many years by several laboratories to mea sure rancidity of food or to follow peroxidative changes in animal metabolism. Reproducibility of the test was good with 20 replicate samples having a coefficient of variation of 4.1^. This assay has the fur ther advantage that malonaldehyde mea surements can be done on either the steam distillate or directly on an acid extract of meat. Vyncke (23) also has reported sim ilar results. Steam distillation of TCA ex tract proved useful in the case of vege tables whose variety of colors interfered with a direct assay. In recent years, the colorimetrie assay has been questioned as possibly being non specific. In this study, we have purified the TBA derivative of malonaldehyde through a series of extractions and further identi fied the TBA derivative through two types of gas chromatography and through thin layer chromatography. Attempts to fur ther identify the TBA derivative through mass spectrometry were not successful. The TBA derivative of malonaldehyde was not volatile at 350°which is the upper limit of attainable column temperature for mass spectral analysis. Pure malonaldehyde is unstable in air and is easily destroyed, but malonalde hyde in beef and other food was quite stable even after cooking. Malonaldehyde may combine with other chemical com ponents in food such as protein to form some very stable compounds. Beef seemed

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consistently to have high levels of malonal dehyde. Even though beef has mainly saturated fat, beef is aged for intervals up to several weeks. Malonaldehyde had ample time to form from the breakdown of unsaturated fat. Some differences in malonaldehyde levels among the various cuts of beef may be due to differences in aging time and handling of the individual cows after slaughter. All of the beef was purchased from local supermarkets and was non-rancid. Pork on the other hand had lower levels of malonaldehyde. It is likely pork is handled faster because of its greater content of unsaturated fat in order to avoid off-flavor odors. Foods such as canned fruit and vegetables had little or no malonaldehyde. The air tight can, natural antioxidants or the skin of the fruit or vegetable may keep the unsatu rated fat from oxidation to malonaldehyde. Several reports have elucidated the harm ful effects of experimental animals eating peroxidized food. Cutler and Hayvvard (3) have summarized the effects of ox idized unsaturated fatty acids in rats: these include damage to the intestinal mucosa with necrosis; edema; increased cytoplasmic vacuoles; inhibition of enzyme systems; oxidation of sulphydryl com pounds; malabsorption syndrome; de creased body weight gain; and an im paired absorption of fat and an increased caloric requirement. Cutler and Schneider (4) have reported an increase in the in cidence of mammary tumors induced by 7,12-dimethylbenzanthracene among rats and mice receiving oxidized linoleic acid in their diet. A disorder of lipid peroxidation in hu mans is characterized by Batten's disease (5). The brains of these patients accumu late a ceroid pigment "age" with pigment. some similari ties to the lipofuscin Chio and Tappel (6) synthesized and charac terized fluorescent products derived from malonaldehyde and amino acids. The Schiff-base products of malonaldehyde crosslinked with primary amino groups of amino acids, proteins, nucleic acids, and their bases, or phospholipids, have fluo rescence properties similar to lipofuscin. Lipofuscin is thought to increase in cells during the aging process (24). Feeding oxidized corn oil to groups of 40 male Charles River rats resulted in an

MALONALDEHYDE

increased number of animals with focal myocarditis and focal fibrosis of the heart (7). Some indications are that malonaldehyde may exist in vivo in older human red cells.1" The source of the malonaldehyde is not known. Malonaldehyde was a good initiator of the carcinogenic process on mouse skin (1). Fifty-eight percent of the mice had tumors after being initiated by 6 mg of malonaldehyde and then promoted by croton oil. Malonaldehyde was a weak complete carcinogen on mouse skin, and also produced several liver carcinomas and a rectal carcinoma. Malonaldehyde was also a mutagen in Ames test system which correlates well with carcinogenicity (2). Part of the mutagenicity and initiat ing activity of malonaldehyde may be due to its reactivity with DNA (25) resulting in genetic damage. Malonaldehyde levels have been mark edly reduced in meat by addition of antioxidants, NaNO- and anaerobic storage (26). LITERATURE

CITED

1. Shamberger, R. J., Andreone, T. L. & Willis, C. E. (1974) Antioxidants and cancer. IV. Malonaldehyde has initiating activity as a carcinogen. J. Nat. Cancer Inst. 53, 17711773. 2. Mukai, F. H. & Goldstein, B. D. (1976) Mutagenicity of malonaldehyde, a decompo sition product of polyunsaturated fatty acids. Science 191, 868-869. 3. Cutler, M. G. & Hayward, M. A. (1974) Effect of lipid peroxides on fat absorption and folie acid status in the rat. Nutr. Metabol. 16, 87-93. 4. Cutler, M. G. & Schneider, R. (1973) Mal formations produced in mice and rats by oxidized linoleate. Fd. Cosmet. Toxicol. 22, 935-942. 5. Siakotos, A. N., Koppang, N., Youmans, B. S. & Bucana, C. (1974) Blood levels of a-tocopherol in a disorder of lipid peroxidation: Batten's disease. Am. I. Ctin. Nutr. 27, 1152-1157. 6. Chio, K. S. & Tappel, A. L. (1969) Syn thesis and characterization of the florescent products derived from malonaldehyde and amino acids. Biochemistry 8, 2821-2827. 7. Kaunitz, H. & Johnson, R. E. (1973) Ex acerbation of heart and liver lesions in rats by feeding of various mildly oxidized fats. Lipids 8, 329-336. 8. Sinnhuber, R. O. & Yu, T. C. (1958) 2Thiobarbituric method for the measurement of rancidity in fishery products. I. The quan'" Goldstein. K. I). & MtDonagli, E. M. (1970) Spectrotlmireseent détection of in vivo lipid peroxldatimi in patients receiving dapsone. Clin. Res. 23, 1274 A.

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titative determination of malonaldehyde. Food Technol. 12, 9-12. 9. Kurkhanova, V. M. & Onekhova, N. V. (1971) Determination of thiobarbituric acid number in fish meat. Issled Technol. Ryb: 73-78. 10. Koning, A. M. & Silk, M. H. (1963) The 2-thiobarbituric acid reagent for determina tion of oxidative rancidity in fish oils. J. Am. Oil Chemists Soc. 40, 167-169. 11. Sinnhuber, R. O. & Yu, T. C. (1968) Char acterization of the red pigment in the 2-thio barbituric acid determination of oxidative rancidity. Food Res. 23, 626-633. 12. Holland, D. C. (1971) Determination of malonaldehyde as an index of rancidity in nut meats. J. Assoc. Offic. Anal. Chem. 54, 1024-1026. 13. Braddock, R. J. & Petrus, D. R. (1971) Malonaldehyde in aqueous orange juice es sences. J. Food Sci. 36, 1095-1097. 14. Arya, S. S. & Ninnala, N. (1971) Deter mination of free malonaldehyde in vegetable oils. J. Food Sci. Technol. 8, 144-180. 15. Sedlocek, B. (1964) Rancidity of fats by ultraviolet spectrophotometry and other meth ods. Nagrung 8, 176-187. 16. Chow, L. & Watts, B. M. (1969) Origin of off-odors in frozen green beans. Food Tech nol. 23, 973-974. 17. Downey, W. K. (1969) Lipid oxidation as a source of off-flavor development during the storage of dairy products. J. Soc. Dairy Tech nol. 22, 154-162. 18. Patton, S. & Kurtz, G. W. (1951) 2-Thiobarbituric acid as a reagent for detecting milk-fat oxidation. J. Dairy Sci. 34, 669-674. 19. Pein, G. (1964) Distillation method for the determination of the shelf-life of whole grain products. Brot. Gebaeck. 18, 12—14. 20. Hutchins, B. K., Liu, T. H. P. & Watts, B. M. ( 1967 ) Effects of additives and refrigeration on reducing activity, metmyoglobin and malonaldehyde of raw ground beef. J. Food Sci. 32, 214-217. 21. Zipser, M. W. & Watts, B. M. (1962) A modified 2-thiobarbituric acid method for the determination of malonaldehyde in cured meats. Food Technol. 16, 102-104. 22. Tarladgis, B. G., Watts, B. M., Younathan, M. T. & Dugan, L. Jr. (1960) A distilla tion method for the quantitative determina tion of malonaldehyde in rancid foods. J. Am. Oil Chemists Soc. 37, 44-48. 23. Vyncke, W. (1975) Evaluation of the di rect thiobarbituric acid extraction method of determining oxidative rancidity in mackerel (Scomber scombrus L.) Fette Seifen An strichmittel 77, 239-240. 24. Miquel, J., Tappel, A. L., Dillard, C. J., Herman, M. M. & Bensch, K. G. (1974) Fluorescent products and lysosomal compo nents in aging Drosophilia melanogaster. J. Gerontology 29, 622-637. 25. Brooks, B. R. & Klamerth, O. L. (1968) Interaction of DNA with bifunctional alde hydes. Eur. J. Biochem. 5, 178-182. 26. Green, B. E. & Price, L. G. (1975) Oxida tion-induced color and flavor changes in meat. Ag. Food Chem. 23, 164-167.

Malonaldehyde content of food.

Malonaldehyde Content oÃ-Food1 RAYMOND J. SHAMBERGER, BARBARA A. SHAMBERGER ANDCHARLES E. WILLIS Department of Biochemistry, The Cleveland Clinic Fou...
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