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Influence of oxidized deep-frying fat and iron on the formation of food mutagens in a model system
Fd Chem. Toxic. Vol. 31, No. 12, pp. 971-979, 1993
0278-6915/93$6.00+ 0.00 Copyright © 1993PergamonPress Ltd
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I N F L U E N C E OF O X I D I Z E D D E E P - F R Y I N G F A T A N D I R O N ON THE F O R M A T I O N OF F O O D M U T A G E N S IN A M O D E L SYSTEM M. JOHANSSON* and M. J.~GERSTAD Department of Applied Nutrition and Food Chemistry, Chemical Center, University of Lund, PO Box 124, S-221 00 Lund, Sweden (Accepted 15 June 1993)
Abstract--The effects of oxidized fats, iron and tocopherol content on the yield and species of mutagenic heterocyclic amines were studied using a model system. A mixture of glycine (0.9 mmol), creatinine (0.9 mmol) and glucose (0.45 mmol) was heated for 10 and 30 min at 180°C, with the addition of iron and fats. The mutagens formed were identified and quantified using HPLC. (2-Amino-3-methylimidazo[4,5-f]quinoxaline) (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) and 2-amino-3,4,8trimethylimidazo[4,5-f]quinoxaline(4,8-DiMelQx) were formed in the model mixtures. The addition of iron (FeSO4) or oxidized fats to the model system did not affect the species of food mutagens formed, but the iron addition more than doubled the amount of MelQx. The oxidation status of the fat added to the model system had little effect on the formation of MelQx. The fat content was shown to affect the mutagen formation significantly,especiallyafter heating for 30 min. No difference in yield of MelQx was observed in the presence of tocopherol and tocotrienol at naturally occurring concentrations.
(150-250°C) (J/igerstad et aL, 1983 and 1991). The imidazo part of the molecule is assumed to be formed Frying or broiling of meat and fish products at high from creatinine, transformed from creatine by heattemperatures may generate several mutagenic hetero- ing. The imidazo moiety reacts with the quinocyclic aromatic amines, these being either amino- line/quinoxaline part, formed from Maillard reaction imidazoazaarenes or amino acid pyrolysates (Felton products, such as pyridines, pyrazines and Strecker and Hatch, 1986; Sugimura and Sato, 1983). The aldehydes. The suggested precursors behind the foraminoimidazoazaarenes have an imidazo group mation of the aminoimidazoazaarenes have been linked to either a quinoline [e.g. MelQ (2-amino- confirmed in model experiments (J/igerstad et al., 3,4-dimethylimidazo[4,5-f]quinoline)], a quinoxaline 1991). [e.g. MelQx (2-amino-3,8-dimethylimidazo[4,5-f]Lipids have been demonstrated to enhance the quinoxaline)] or a pyridine [e.g. PhlP (2-amino-1- yield of pyrazines, pyridines and Strecker aldehydes methyl-6-phenylimidazo[4,5-b]pyridine)] and are in model systems (Arnoldi et al., 1987 and 1990; suggested to be formed from creatinine, free amino Buttery et aL, 1977; Kawamura, 1983; Parihar et al., acids and monosaccharides naturally occurring in 1981; Watanabe and Sato, 1971a,b). One may thereprotein-rich foods, at normal cooking temperatures fore expect the yield of heterocyclic amines to be increased by lipids. This was also shown in a previous study conducted at our laboratory (Johansson et al., *To whom correspondence should be addressed. 1993) by heating the precursors of the heterocyclic Abbreviations: BHA=butylated hydroxyanisole; BHT= butylated hydroxytoluene; DCM=dichloromethane; amines in a model system containing oils such as corn 4,7-DiMelQx = 2-amino-3,4,7-trimethylimidazo[4,5-f]- oil and olive oil. However, heating the precursors in quinoxaline; 4,8-DiMelQx = 2-amino-3,4,8-trimethyl- the presence of glycerol or various fatty acids had imidazo[4,5-flquinoxaline; 5,8-DiMelQx= 2-amino(except in the case of stearic acid) no effect on the 3,5,8-methylimidazo[4,5-f]quinoxaline;7,8-DiMelQx = 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline; yield. IQ = 2-amino-3-methylimidazo[4,5-f]quinoline; IQx = The possibility that lipids, by being oxidized, might 2-amino-3-methylimidazo[4,5-f]quinoxaline; IS = affect the yield of the heterocyclic amines was not internal standard; iso-IQ=2-amino-l-methylimidazosystematically investigated in our previous study [4,5-f ]quinoline; LC = liquid chromatography; MelQ = (Johansson et al., 1993). Lipid oxidation is known 2-amino-3,4-dimethylimidazo[4,5-f]quinoline;MelQx = to produce free radicals (Eriksson, 1987) and free 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; MS= mass spectrometry; PG = n-propylgallate; PHIP = radical reactions have been proposed to increase 2-amino- l-methyl-6-phenylimidazo[4,5-b]pyridine; the formation of heterocyclic amines (Barnes and SIM =selected ion monitoring; T B H Q = t e r t - b u t y l hydroquinone; 4,7,8-TriMelQx = 2-amino-3,4,7,8-tetra- Weisburger, 1984; Namiki and Hayashi, 1983; methylimidazo[4,5-f]quinoxaline; TLC = thin-layer Yoshida and Mizusaki, 1985; Yoshida et al., 1986). chromatography. Furthermore, lipid hydroperoxides might produce INTRODUCTION
971
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M. JOHANSSON and M. J~oERSTAD
aldehydes, which could react with amino acids to give Schiff base adducts in analogy to the Maillard reaction (Gardner, 1979). Also, through autoxidation of lipids, carbonyl compounds may be formed that can react with amino compounds to produce browning products of high molecular weight, as reported by Kawamura (1983). The main purpose of this study was to investigate whether the oxidation of lipids affects the yield and principles of heterocyclic amines in a model system. Furthermore, this study was performed to investigate the effect of iron and the combination of oxidized fat and iron on the formation of heterocyclic amines. Both lipids and iron compounds are present during the cooking of meat and fish, in native form (fat, haem iron) as well as in added forms (such as oil, margarine and iron by leaching from the cooking equipment). Iron is known to act as a pro-oxidant in lipid oxidation during the formation of free radicals, which might potentiate the Maillard reaction. On the other hand, unoxidized oil contains tocopherols, which might have a protective effect in their role as free radical scavengers. Thus, if free radicals have any impact on the yield of heterocyclic amines, vitamin E could have a protective role. This was evaluated by comparing the effects of unoxidized oil with those of oxidized oil. Studies of factors behind the formation of heterocyclic amines produced during the cooking of meat and fish are motivated by the fact that such compounds, when tested in rodent bioassays, have been shown to be multipotent carcinogens (for a review see Ohgaki et al., 1991). Dietary fats are also known to increase the risk of cancer, according to several hypotheses that are under investigation (Committee on Diet, Nutrition, and Cancer, 1982; Henderson, 1990; Tomatis, 1990; Wilett el al., 1990). None of them include the possibility that dietary fats might promote the formation of food carcinogens such as the heterocyclic amines, which is an aspect that is investigated in the study described here. MATERIALS AND METHODS
Chemicals. All commercially available chemicals were of analytical grade. Water was taken from a Milli-Q water purification system (Millipore). All solvents [acetonitrile, methanol, dichloromethane (DCM), heptane and di-isopropylether] were purchased from Merck A G (Darmstadt, Germany). [~4C]MelQx [10 mCi/mmol, 98% radiochemical pure according to nuclear magnetic resonance spectral analysis and thin-layer chromatography (TLC)], synthetic MelQx standard solution (100mg/ml, estimated to be greater than 98% pure by TLC and melting point) and iso-IQ (2-amino-l-methylimidazo[4,5-f]quinoline) were purchased from Toronto Research Chemicals (Toronto, Canada). Synthetic IQ (2-amino-3-methylimidazo[4,5-f]quinoline), MelQ,
MelQx, 4,7-DiMeIQx (2-amino-3,4,7-trimethylimidazo[4,5-J]quinoxaline), 4,8-DiMeIQx (2-amino-3,4,8trimethylimidazo[4,5-f]quinoxaline), 5,8-DiMeIQx (2-amino-3,5,8-methylimidazo[4,5-f]quinoxaline), 7,8DiMeIQx (2-amino-3,7,8-trimethylimidazo[4,5-J]quinoxaline) and 4,7,8-TriMeIQx (2-amino-3,4,7,8tetramethylimidazo[4,5-f]quinoxaline) were kind gifts from Professor Kjell Olsson and Dr Spiros Grivas (Department of Chemistry and Molecular Biology, Uppsala, Sweden). IQx (2-amino-3-methylimidazo[4,5-f]quinoxaline) was a kind gift from Mark G. Knize (Lawrence Livermore Laboratory, CA, USA). Ecoscint and Ecoscint A scintillation fluids were obtained from National Diagnostics (Somerville, N J, USA). Dimodan OT (distilled monoglyceride) was obtained from Grindsted (Denmark). Tocopherol standard (~, 7, f-isomer kit, 50 mg per ampoule) was obtained from Merck AG (Darmstadt, Germany). Iron(II)sulfate heptahydrate (FeSO4) was from Merck AG (Darmstadt, Germany). Corn oil was purchased in a local shop. Five partially oxidized samples (nos 1-5) of a deep-frying fat were manufactured by Karlshamns AB (Sweden). The fat consisted of palm oil and hydrated rapeseed oil, which is a common combination in commercially used deep-frying fats. The composition of the fat was as follows: C~4,, (0.6%): Ci6:0 (25.6°/o); Cis:0 (9.1°/oL Cl~:l (54.9%): Ci82 (7.4%); C20:0 (0.6%) and C20:1 (0.6%). A Foodoil Sensor, which measures the dielectric constant of the fat and correlates it to the degree of oxidation, was used to select five partially oxidized samples of the fat (1 =unused fat; 2 =half-used fat; 3 = near upper level for usage; 4 = more oxidized than the upper level for usage according to the Foodoil Sensor, but amounts of polar compounds not exceeding 3 0 ° . which is the recommended upper level for usage according to the Swedish National Food Administration (SLV FS 1990:); 5 = o v e r upper level for usage, exceeding levels of polar compounds and according to the Foodoil Sensor). The samples of oxidized fat were analysed for anisidine value (IUPAC 2.504), peroxide value (AOCS Cd-8-53 modified), free fatty acids, amounts of polar compounds and tocopherol and tocotrienol content (1UPAC 2.432) by Karlshamns AB. The oxidation status of the five partially oxidized fats is shown in Table 1. Model system. The precursors --creatinine (0.9 mmol), glycine (0.9 mmol) and glucose 0.45 mmol)--were dissolved in Milli-Q water, before being heated in closed test-tubes at 180'C for 10 and 30 rain, as described earlier (Johansson et al., 1993). H202 (100#i, 30%) was added and oxygen passed through the mixture for 1 min in order to initiate free radical reactions. The study was performed in three different experiments. In the first, the precursors were dissolved in 2.5 ml Milli-Q water; in the second and third experiments 0.5 g (20%) and 1.0 g (40%) respectively, of one of the five deep-frying fat samples were melted with 5 nag Dimodan OT before addition
Oxidized fats, iron and food mutagens of the precursors dissolved either in 2.0 or 1.5ml Milli-Q water. The solutions were shaken until an emulsion appeared, before heating as above. The experiment was repeated with the addition of FeSO4 (0.09 mmol) in the model system. To ensure that D i m o d a n O T had no effect on mutagen formation, creatinine, glycine and glucose were dissolved in Milli-Q water and heated for 10 rain at 180°C with and without the addition of D i m o d a n OT. Another experiment was performed by heating creatinine, glycine, glucose, corn oil and D i m o d a n O T dissolved in Milli-Q water for 10 min at 180°C without H202 or oxygen. For the determination of vitamin E in the heated mixtures, creatinine, glycine, glucose and D i m o d a n O T were heated for 10 and 30min at 180°C with deep-frying fat number 1 after treatment with H202 and oxygen, as described above. The crude fat (no. 1) was also heated for 10 and 3 0 m i n at 180°C. All experiments were performed in triplicate. Purification. The heated samples were spiked with 8.1 nCi [14C]MelQx serving as an internal standard (IS) before purification. The samples were extracted according to the method of Gross (1990) with some minor modifications (Johansson et al., 1993). After purification, the samples were dissolved in 100/al methanol. Aliquots (50%) of the samples were used for the determination of [14C]MelQx (IS) recovery, using a liquid scintillation counter (1219 Rackbeta LKB Wallac, Sweden). H P L C fractionation. The sample volume was reduced to dryness under nitrogen and finally dissolved in 125 #1 H P L C buffer A (see below). Aliquots (15/al) of the sample were injected into a Varian 9010 Liquid C h r o m a t o g r a p h (LC), with a photo-diode array UV detector (Varian 9065, Polychrom), equipped with an O D S 80 column (ToyoSoda TSK gel TM, 250 x 4.6 mm i.d., 5 ~tm particle size, Varian, Stockholm, Sweden) and eluted with a mobile phase of 0,01 M triethylamine in water adjusted with acetic acid to p H 3.2 (A) and p H 3.6 (B), and acetonitrile (C). Gradients of 5-15% C in A for 10min, then 15-25% C in B for 10rain, and finally 25-55% C in B for 5 min were used. The flow rate was 1.0 ml/min and the effluent was monitored at 263 nm. In some experiments, fractions were collected every 30 sec
973
between 8 and 30 min and diluted before the assay of mutagenic activity. Mutation assay. The mutagenic activity of the fractions was tested as described by Ames et al. (1975) using Salmonella strain T A 98 with the addition of 0.5 ml S-9 mix containing 5% chlorophene-induced rat liver per plate (Maron and Ames, 1983). The fractions were tested at one dose (25 pl out of a total volume of 1 ml). Synthetic M e l Q x was used as a positive control (50,000 revertants//zg). The colonies were counted in an automated colony counter (Bio Tran II, New Brunswick Scientific Co, Inc., N J, USA). The spontaneous reversion rate was 30-35 revertants/plate. A fraction was considered mutagenic if it induced a number of revertants that was twice the background level. H P L C identification and quantification. The mutagens produced in the model system were fractionated using HPLC, as described above. The identities of the mutagens were established by comparing the retention times of the mutagenic peaks, according to the Ames test, with the retention times of synthetic compounds, namely IQ, iso-IQ, IQx, MelQ, MelQx, 4,7-DiMelQx, 4,8-DiMelQx, 5,8-DiMelQx, 7,8D i M e l Q x and 4,7,8-TriMelQx, obtained under the same conditions. In addition, some samples were also spiked with synthetic compounds before injection. Diode array UV spectra of the peaks were compared with diode array UV spectra of synthetic compounds obtained under the same conditions. The mutagens were quantified by comparing the peak area of the H P L C chromatographed sample with the area of a known amount of standard. The amount was corrected for the recovery of [a4C]MelQx. M S identification. Two of the mutagens formed in the model system were identified earlier as MeIQx and 4,8-DiMeIQx using mass spectrometry (MS) (Johansson et al., 1993). The third mutagenic fraction isolated from a heated model mixture (creatinine, glycine and glucose heated in 2.5 ml Milli-Q water for 10min at 180°C) was identified using L C - M S with selected ion monitoring (SIM). The mass spectrum obtained was compared with that of synthetic IQx. Determination o f vitamin E. The tocopherol and tocotrienol content (~-, 7- and 6-forms) were determined in the crude fat (no. I), heated crude fat (no. 1)
Table 1. Foodoil Sensor*, peroxide value, free fatty acids, anisidine value, polar compounds, tocopherol and tocotrienol contents in the partially oxidized deep-frying fats (nos I-5) (single determinations) Peroxide Polar Tocopherol (T)/tocotrienol (T3) content (mg/kg) Deep-frying Foodoil value Free fatty Anisidine compound fatt Sensor (mEq/kg) acids (%) value (%) -T#t -T 3 y -T/3' -T3 ~-T/6-T 3 1 re~rence 3.2 0.03 4.0 4.4 160/70 180/90 10/20 2 1.6 6.1 0.2 65.9 13.0 <1 <1 <1 3 3.7 5.2 0.4 87.8 25.1 <1 <1 <1 4 5.2 4.5 0.7 94.9 27.8 <1 <1 <1 5 >6 3,2 1.0 97.9 32.6 <1 <1 <1 *The Foodoil Sensor measures the dielectric constant of the fat and correlates it to the degree oif oxidation. tl = unused fat; 2 = half-used fat; 3 = near upper levelfor usage; 4 = more oxidized than the upper level for usage according to the Foodoil Sensor, but amounts of polar compounds not exceeding30% (the recommended upper levelfor usage according to the Swedish National Food Administration).
974
M. JOHANS.,tK)Nand M. JJ~GERSTAD
E t-
@ ¢q O O
O
[q 8
400 200 0
1
5
10
15
20
25
min
Fig. 1. UV absorbance at 263 nm and mutagenic activity in (revertants/fraction) in fractions collected from HPLC fractionation of a mixture of creatinine, glycineand glucose heated at ! 80°C for 10 min. Peaks at retention times 11, 15 and 18min correspond to 2-amino-3-methylimidazo[4,5-f]quinoline(IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMelQx), respectively.
and in model systems containing fat (no. 1) and precursors, heated for 10 and 30 min at 180°C. The vitamin E forms were quantified using an HPLC (Varian 5000 Liquid Chromatograph) with a fluorescence detector (Shimadzu Spectrofluorophotometer, RF-540) set at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. The HPLC was equipped with a Lichrosorb Si-60-5 column (Chrompack 250 x 4.6 mm i.d.), and eluted isocratically with a mobile phase of heptane:diisopropylether (91:9), at a flow rate of 2.5 ml/min (H~kansson et al., 1987). 20/~1 of the samples (only the fat phase was taken from the heated model mixtures) dissolved in heptane (200mg sample/ml heptane for the crude samples, 400 mg sample/ml heptane for the model mixtures) and standard solutions (ct-, y- and 6-tocopherols, 10#g/ml heptane) were injected onto the HPLC column. Statistical evaluation. The differences between the samples ( n > 12) were evaluated statistically with Student's t-test using a PC version of Minitab.
RESULTS Mutagens produced in the model system Three major mutagenic compounds were formed in the heated model mixtures, according to the results of the Ames test on fractions from HPLC. The same mutagenic pattern was observed for all heated mixtures containing any of the fats and regardless of whether FeSO4 was added or not. In Fig. 1 the mutagenic activity from a heated mixture and a corresponding chromatogram are shown. Two of the mutagenic compounds were earlier identified as MelQx and 4,8-DiMelQx using MS. In the present study, their identities were confirmed by comparing their UV spectra with the UV spectra of synthetic references. The third mutagenic fraction corresponded to synthetic IQx in retention time and UV spectrum. Furthermore, the peak co-eluted with synthetic IQx when fractionated on HPLC. The presence of IQx was confirmed using LC-MS with SIM. In Fig. 2 an LC-MS chromatogram of the fraction containing IQx and synthetic IQx is shown.
975
Oxidized fats, iron and food mutagens
Effects of oxidized fats and iron on the formation of MelQx The quantitative results from the HPLC analysis of the heated mixtures are shown in Fig. 3. The amounts were corrected for [14C]MeIQx recovery, which varied between 52 and 97%.
Heating the precursors (creatinine, glycine, glucose, H202 and oxygen) in the absence of fats at 180°C for 10 and 30 min produced 3.4 and 10.4 nmol MeIQx/mmol creatinine, respectively, if no FeSO4 was present and 10.6 and 17.2nmol MeIQx/mmol creatinine, respectively, in the presence of FeSO4. The amount of MeIQx formed was significantly increased
11:57 100
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50
3:20
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Fig. 2. Plasma-spray mass spectra. Single-ion recording of the protonated molecular ion (MH+). Top: mutagenic fraction from a heated (180°C/10 min) mixture of creatinine, glycine and glucose. Bottom: synthetic IQx.
mL~
976
M . JOHANSSON a n d M . J~GERSTAD
No fat 17,- - "1 . . . . . . . . . Fat no 1 20% I . . . . . . Fat n o 2 2 0 %
I...~
] ....... ..........
Fat n o 3 20% r. 7 ~ T . '
.....
~--.
Fat no 4 20% k ¢ ¢ ~ f i t . . . . . Fat no 5 20% 7 Fat no 1 4 0 %
7-7'' 1 .......
Fat no 2 4 0 %
¢ ....
Fat n o 3 4 0 % I . . . . . Fat no 4 4 0 % Fat n o 5 4 0 %
_
. . . . . . . . . . . . . . ~. . . . . . . .
--
t .........
............. ~ I 0
....... I 5
I 10
I 15
I 20
I 25
30
nmol MelOx/mmol creatnine Fig. 3. Amounts of MelQx (nmol MelQx/mmol creatinine) formed in model systems containing creatinine, glycine, glucose, deep-frying fat (nos 1 5), Dimodan OT, H202 and oxygen, with and without the addition of FeSO4. All values are means of three measurements: 71, 10 min at 180°C without iron; [], 10min at 180°C with iron; D, 30 min at 180°C without iron; II, 30min at 180°C with iron. (P < 0.001) when the model mixture was heated at 180°C for 30min compared with 10min, both with and without iron and any of the fat samples. The degree of oxidation of the fat had small effects on the formation of MelQx in the model system. As can be seen in Fig. 3, no consistent trend was demonstrated between the yield of MelQx and the degree of oxidation. When comparing all mixtures containing any of the fats, significantly higher (P <0.05) amounts of MelQx were formed in model mixtures containing 40% fat than with 20% fat. Comparing the MelQx content in model mixtures without fat, and mixtures with 20% or 40% of all fat samples, showed significant differences (P < 0.05) only when an iron-containing model mixture was heated for 30 min with 20% fat or when a mixture with or without iron was heated for 30 min with 40% fat. The addition of iron (FeSO4) significantly increased the formation of MelQx more than two-fold (P < 0.001), when the mixture was heated for both 10 and 30min at 180°C. Similar amounts of MelQx were formed both in a mixture containing Dimodan OT, heated for 10 min at 180°C, and in a mixture containing no emulsifier, 6.7 and 4.0 nmol MelQx/mmol creatinine, respectively. Thus, neither the addition of a monoglyceride as an emulsifier nor the addition of H202 and oxygen in the model mixtures had any quantitative effect on mutagen formation (P > 0.05). V i t a m i n E content
Only sample no. 1 contained natural tocopherols and tocotrienols, whereas all vitamin E activity had been destroyed in the more oxidized samples (nos 2-5) (Table !). The tocopherol and tocotrienol content in crude unheated fat (no. 1), heated fat (no. 1) and in a heated model system containing fat (no. 1) is shown in
Table 2. As can be seen, the ~-tocopherol and 7tocotrienol contents were affected most, but were similar in the model mixtures and the heated fat. The 7-tocopherol and ),-tocotrienol contents were lower when the fat or model system was heated for 30 min than when it was heated for 10 min. The 6-tocopherol and 6-tocotrienol contents were not affected by heating in any of the cases, being 10 and 20 mg/kg for 6-tocopherol and 6-tocotrienol, respectively. DISCUSSION
Three major mutagenic peaks were identified in all the heated mixtures. Thus, the addition of fat, oxidized fat or iron did not affect the principles of the heterocyclic amines. This has been shown previously (Johansson et al., 1993) with the addition of different fatty acids and oils in a similar model system. The mutagenic peaks corresponded to IQx, MeIQx and 4,8-DiMelQx, respectively. The formation of MelQx, 4,8-DiMelQx and also 7,8-DiMelQx from creatine, glycine and glucose in model systems in the absence of fat, has been reported earlier (Jiigerstad et al.,
Table 2. Tocopherol (T) and tocotrienol (T 3) content (mg/kg) in unheated crude fat (no. 1)*, heated fat (no. 1) and in heated model mixtures containing fat (no. 1) (means of two determinations) ~-T/0:-T 3
7-T/y-T 3
6 -TI6-T 3
Unheated fat
190/70
240/I 10
10/20
Heated fat (180 C / 1 0 min)
110/30
190/80
10/20
Heated fat (180 C/30 min)
40/10
110/50
10/10
Model system (180~'C/10 min)
110/30
170/80
10/20
Model system (180°C/30 min)
80/20
150/70
10/20
Sample
*As in Table 1.
Oxidized fats, iron and food mutagens 1986 and 1991; Negishi et al., 1984; Skog and J/igerstad, 1990). The formation of IQx has only previously been reported in a dry-heating experiment involving serine and creatinine (Knize et al., 1988). However, IQx might have been formed before, but escaped detection by being lost during the commonly used XAD-2 purification (Felton and Hatch, 1986). Both MeIQx and 4,8-DiMeIQx are usually formed in fried beef (Felton and Knize, 1990). IQx has only been found in a fried meat emulsion with added creatinine (Becher et al., 1988). The yield of MeIQx was shown to increase significantly with increased heating time (10-30 min), lipid concentration (20--40%) and by adding FeSO, (the Fenton reaction). The addition of an emulsifier, Dimodan OT, did not affect the yield, neither did the presence of hydrogen peroxide and oxygen. The results support those of our previous study (Johansson et al., 1993) also showing increases in the yield of MeIQx in the presence of oil and with increased heating time. The observation that deep-frying oil affected the yield of MeIQx is in agreement with previous studies demonstrating an enhancing effect of lipids on the formation of Maillard reaction products, e.g. pyrazines, pyridines and Strecker aldehydes (Arnoldi et al., 1987 and 1990; Buttery el al., 1977; Kawamura, 1983; Parihar et al., 1981; Watanabe and Sato, 1971a,b). As these products are assumed to be precursors of the IQ compounds (J/igerstad el al., 1983 and 1991), an increase in their formation should be associated with a corresponding increase in the yield of IQ compounds. It was also of interest to investigate whether oxidized lipids had any specific effect on the yield of the heterocyclic amines. As each oxidized oil sample tested was investigated only in triplicate, it was not possible to evaluate the results statistically. However, although there were some differences in mutagen formation between the fat samples, there seemed not to be any consistent trend with the degree of oxidation. This might perhaps be attributable to the addition of hydrogen peroxide and oxygen to all samples regardless of degree of oxidation, thereby possibly masking differences in their effects on the yield of MeIQx. However, in a separate experiment, we found no effect on the yield of MeIQx by adding oxygen and hydrogen peroxide to the model system. The reason why all the samples were treated with hydrogen peroxide and oxygen was to create more favourable conditions for the oxidized oils to produce free radicals during heating in the model system. This experimental design does not allow any conclusions to be drawn on the effects of oxidized fat p e r se on the formation of MeIQx. However, the results seem to indicate that free radicals produced from the fat oxidation catalysed by H202 and oxygen did not have any marked influence on the yield of MeIQx. Interestingly, the Fenton reaction (Fe :+ + H:O2--*Fe3+ + O H - + O H ' ) doubled the yield of MeIQx (P < 0.001), both when the mixtures were
977
heated for 10 and for 30min at 180°C. The iron catalyses extremely reactive free radicals, such as lipidperoxy radicals. The doubling of the MeIQx yield by the Fenton reaction was seen both in the presence and absence of fats. Iron has previously been reported to enhance the formation of food mutagens in fried beefburgers. Barnes and Weisburger (1984) added 10 ppm Fe 2+ and Fe 3+ to meat before frying and reported a doubling in mutagenic activity. The authors suggested that the catalytic effect of iron ions was related to their capacity to accelerate free radical reactions. Taylor et al. (1986) reported that FeSO4 increased the formation of IQlike compounds by nearly doubling the mutagenic activity in a model mixture consisting of creatine phosphate and tryptophan. Moreover, the findings reported by Parihar et al. (1981) indicate that iron and copper ions catalyse the formation of pyrazines and pyridines when heating oxidized oil and amino acids in the absence of sugar. Thus, the mechanism by which free radicals might affect the yield of MeIQx is probably through an increase of precursors such as pyrazines and pyridines, although other mechanisms such as activation of the reactants cannot be excluded. The enhancing effect of iron is very interesting since there are two possible iron sources during the frying of meat--haem-iron released through the denaturation of myoglobin present in animal muscle, and by leaching from the cooking equipment itself. The haem-iron source is difficult to influence, but iron leaching from cooking equipment might be reduced by using iron-free material such as teflon [poly(tetrafluoroethene)]. The enhancing effect of free radical reactions on the yield of MeIQx seems to be supported by the Fenton reaction, but the results of this study indicate that lipid oxidation without catalysts is insufficient to induce enough free radicals to affect the yield of MeIQx. Vitamin E is a natural lipid antioxidant known to be an effective free radical scavenger (Burton and Ingold, 1981). The least oxidized fat sample (no. 1) was the only sample that contained vitamin E (640 ppm); however, this sample produced a yield of MelQx similar to those in samples lacking vitamin E. During the heating of oil in the model system, vitamin E was partly lost, but between 20 and 30% of the ~t-forms resisted heating and more than half of the ~and c~-forms were present after 30 min heating. Thus, the presence of vitamin E in sample no. 1 seemed not to have any pronounced inhibitory effect on the yield of MeIQx, regardless of whether iron was present or not. If free radicals are an important contributory mechanism for the formation of heterocyclic amines, the sample containing natural vitamin E would have exhibited a lower yield of MeIQx than the other oxidized deep-frying oils, but this was not the case. Perhaps the concentration of vitamin E needs to be considerably higher in order to exert inhibitory effects. Studies have been performed by Pearson et al.
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M. JOHANSSONand M. J~GERSTAD
(1992) and Chen et al. (1992) on the effect of different natural and synthetic antioxidants during the frying of beef. Vitamin C (100ppm), vitamin E, B H A (butylated hydroxyanisole), P G (n-propylgallate) and T B H Q (tert-butyl-hydroquinone) (0.1% of fat content) decreased the formation of IQ compounds by about 50-70%. In contrast BHT (butylated hydroxytoluene) (0.1% of fat content) enhanced the formation of 4,8-DiMelQx by 12%. The amounts of the other two heterocyclic amines identified, 4,8-DiMelQx and IQx, were roughly estimated to be 1-2 and 9 nmol/mmol creatinine, respectively. Neither of these two compounds seemed to be affected by heating time, lipid concentration, degree of oxidation or by the Fenton reaction. However, since the quantification of these two compounds was not based on recovery corrections performed by spiking the samples, it is not possible to draw any definite conclusions about these two IQ compounds regarding yield-affecting factors such as lipids and free radicals. In conclusion, the study described here demonstrates a significant chemical effect of lipids on the yield of MelQx, catalysed by iron and probably mediated by the Maillard reaction. These findings add further yield-affecting factors to the formation of the heterocyclic amines. In a previous study, we have shown that lipids affected the amount of mutagenic activity by creating more efficient heat transfer during the cooking of meat loaf (Holtz et al., 1985). Thus, one explanation of the increased formation of heterocyclic amines during the cooking of products high in fat or with added fat could be simply a temperature rise. The model system used here excluded such a possibility, since the heating was performed under strictly controlled conditions. It therefore seems that lipids enhance the formation of IQ compounds both by physical and chemical means. Previous studies reported in the literature have shown lipids to have enhancing effects, no effect or even inhibitory effects (Barrington et al., 1990; Bjeldanes et al., 1983; Knize et al., 1985; Nilsson et al., 1986; Spingarn et al., 1981). In general, these studies do not discuss the yield-affecting mechanisms; furthermore, most of them rely on measurements of mutagenic activity only, and it is not always clear whether the investigators have excluded possible inhibitory effects of certain fatty acids on the Ames test. An aspect that has hitherto escaped attention is whether naturally occurring antioxidants in meat or oils, such as tocopherols or fl-carotene, might inhibit the formation of heterocyclic amines during cooking. This study did not give support the theory that vitamin E at concentrations occurring naturally in deep-fat frying oil has any protective role in this respect, but further research on this topic is necessary. Acknowledgements--We are grateful to Karlshamns AB, Sweden, for manufacturing and analysing the oxidized fat samples, Drs G. A. Gross and L. Fay, Nestl6 Research
Centre (Vers-Chez-les-Blanc, Switzerland) for performing the LC-MS analysis and Mrs Hanna Nilsson and Gunnel Andersson, Department of Applied Nutrition and Food Chemistry, for their excellent technical assistance. The kind gifts of synthetic references from Professor Kjell Olsson and Dr Spiros Grivas (Uppsala, Sweden) and Mark G. Knize (Livermore, CA, USA) are gratefully acknowledged. Salmonella typhinurium strain TA 98, was kindly provided by Professor Bruce Ames, University of Berkeley (Berkeley, CA, USA). This study was supported by the Swedish Cancer Foundation (1824-B92-11XAC) and the Swedish Council for Forestry and Agricultural Research (50.0440/91). REFERENCES
Ames B. N., McCann J. and Yamasaki E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella mammalian-microsomal mutagenicity test. Mutation Research 31, 347-364. Arnoldi A., Arnoldi C., Baldi O. and Ghizzoni C. (1990) Effect of lipids in the Maillard reaction. In The Maillard Reaction. Advances in Life Sciences. Edited by P. A. Finot, H. U. Aeschbacher, R. F. Hurrell and R. Liardon. pp. 133-138. Birkh/iuser Verlag, Basel. Arnoldi A., Arnoldi C., Baldi O. and Griftini A. (1987) Strecker degradation of leucine and valine in a lipidic model system. Journal of Agricultural and Food Chemistry 35, 1035-1038. Barnes W. S. and Weisburger J. H. (1984) Formation of mutagens in cooked foods. VI. Modulation of mutagen formation by iron and ethylenediaminetetracetic acid (EDTA) in fried beef. Cancer Letters 24, 221 226. Barrington P. J., Baker R. S., Truswell A. S., Bonin A. M., Ryan A. J. and Paulin A. P. (1990) Mutagenicity of basic fractions derived from lamb and beef cooked by common household methods. Food and Chemical Toxicology 28, 141 146. Becher G., Knize M. G., Nes 1. F. and Felton J. S. (1988) Isolation and identification of mutagens from a fried Norwegian meat product. Carcinogenesis 9, 247-253. Bjeldanes L. F., Morris M. M., Timourian H. and Hatch F. T. (1983) Effects of meat composition and cooking conditions on mutagen formation in fried ground beef. Journal of Agricultural and Food Chemistry 31, 18-21. Burton G. W. and Ingold K. U. (1981) Autooxidation of biological molecules. 1. The antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in t'itro. Journal of The American Oil Chemists" Society 103, 6472-~477. Buttery R. G., Ling L. C., Teranishi R. and Mon T. R. (1977) Roasted lamb fat: basic volatile components. Journal of Agricultural and Food Chemistry 25, 1227-1229. Chen C., Pearson A. M. and Gray J. I. (1992) Effects of some additives on mutagen formation during frying of ground beef. Proceedings of the 38th International Congress on Meat Science and Technology (ICoMST), 23 28 August 1992, Clermont-Ferrand, France. pp. 475--478. Commitee on Diet, Nutrition and Cancer (1982) In Diet, Nutrition and Cancer. National Academic Press, Washington, DC. Eriksson C. E. (1987) Oxidation of lipids in food systems. In Autoxidation of Unsaturated Lipids. Edited by H. W. Chan. pp. 207 232. Academic Press, London. Felton J. S. and Hatch F. T. (1986) Mutagens in cooked food. Energy Technology Review 1 15. Felton J. S. and Knize M. G. (1990) Heterocyclic amine mutagens/carcinogens in foods. In Handbook of Experimental Pharmacology. Edited by C. S. Copper and P. L. Grover. pp. 471-502. Springer Verlag, Berlin. Gardner H. W. (1979) Lipid hydroperoxide reactivity with proteins and amino acids: a review. Journal of Agricultural and Food Chemistry 27, 220-229.
Oxidized fats, iron and food mutagens Gross G. A. (1990) Simple methods for quantifying mutagenie heterocyclic amines in food products. Carcinogenesis 11, 1597-1603. Henderson M. M. (1990) Correlations between fatty acid intake and cancer incidence. In Health Effects of Dietary Fatty Acids. Edited by G. J. Nelson. pp. 136-149. American Oil Chemists' Society, Champaign, IL. Holtz E., Skj61debrand C., Jagerstad M., Laser Reutersward A. and Isberg P. E. (1985) Effect of recipes on crust formation and mutagenicity in meat products during baking. Journal o f Food Technology 20, 5746. H~tkansson B., J~igerstad M. and Oste R. (1987) Determination of vitamin E in wheat products by HPLC. Journal of Micronutrient Analysis 3, 307-318. Johansson M., Skog K. and Jagerstad M. (1993) Effects of edible oils and fatty acids on the formation of mutagenic heterocyclic amines in a model system. Carcinogenesis 14, 89-94. J/igerstad M., Grivas S., Olsson K., Laser Reutersw~d A., Negishi C. and Sato S. (1986) Formation of food mutagens via Maillard reactions. In Proceeding Monography: Genetic Toxicology of the Diet. Edited by I. Knudse~. pp. 155-167. Progress in Clinical and Biological Research. Vol. 206. Alan R. Liss, Inc., New York. Jagerstad M., Laser Reutersw/ird A., Oste R., Dahlqvist A., Olsson K., Grivas S. and Nyhammar T. (1983) Creatinine and Maillard reaction products as precursors of mutagenic compounds formed in fried beef. In The Maillard Reaction in Foods and Nutrition. ACS Symposium Series 215. Edited by G. R. Waller and M. S. Feather. pp. 507-519. American Chemical Society, Washington DC. J/igerstad M., Skog K., Grivas S. and Olsson K. (1991) Formation of heterocyclic amines using model systems. Mutation Research 259, 219-233. Kawamura S. (1983) Seventy years of the Maillard reaction. In The Maillard Reaction in Foods and Nutrition. ACS Symposium Series 215. Edited by G. R. Waller and M. S. Feather. pp. 3-18. American Chemical Society, Washington DC. Knize M. G., Andresen B. D., Healy S. K., Shen N. H., Lewis P. R., Bjeldanes L. F., Hatch F. T. and Felton J. S. (1985) Effects of temperature, patty thickness and fat content on the production of mutagens in fried ground beef. Food and Chemical Toxicology 23, 1035-1040. Knize M. G., Shen N. H. and Felton J. S. (1988) The production of mutagens in foods. Proceedings o f the Air Pollution Control Association, Dallas, Texas, 19-24 June 1988. pp. 88-130. Maron D. M. and Ames B. N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research 113, 173-215. Namiki M. and Hayashi T. (1983) A new mechanism of the Maillard reaction involving sugar fragmentation and free radical formation. In The Maillard Reaction in Foods and Nutrition, ACS Symposium Series 215. Edited by G. R. Waller and M. S. Feather. pp. 21-46. American Chemical Society, Washington, DC. Negishi C., Wakabayashi K., Tsuda M., Sato S., Sugimura
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T., Saito H., Maeda M. and Jagerstad M. (1984) Formation of 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline, a new mutagen, by heating mixture of creatinine, glucose and glycine. Mutation Research 140, 55-59. Nilsson L., Overvik E., Fredholm L., Levin 0., Nord C.-E. and Gustafsson J.-,~. (1986) Influence of frying fat on mutagenic activity in lean pork meat. Mutation Research 171, 115-121. Ohgaki H., Takayama S. and Sugimura T. (1991) Carcinogenicities of heterocyclic amines in cooked food. Mutation Research 259, 399--410. Parihar D. B., Vasundhara T. S. and Vijayayaghavan P. K. (! 981) Effect of metal ions such as iron and copper on the formation of nitrogen heterocyclic compounds in sesame oil-or-amino acids model systems under frying conditions. Journal of Food Technology 16, 313-319. Pearson A. M., Chert C. and Gray J. I. (1992) Effects of different antioxidants on formation of meat mutagens during frying of ground beef. Proceedings of the 38th International Congress on Meat Science and Technology (ICoMST), 23-28 August 1992, Clermont-Ferrand, France. pp. 567-570. Skog K. and Jagerstad M. (1990) Effects of monosaccharides and disaccharides on the formation of food mutagens in model systems. Mutation Research 230, 263-272. Spingarn N. E., Garvie-Gould C., Vuolo L. L. and Weisburger J. H. (1981) Formation of mutagens in cooked foods. IV. Effect of fat content in fried beef patties. Cancer Letters 12, 93-97. Sugimura T. and Sato S. (1983) Mutagens-caracinogens in food. Cancer Research (Suppl.) 43, 2415s-2421s. Taylor R. T., Fultz E. and Knize M. (1986) Mutagen formation in a model beef supernatant fraction. IV. Properties of the system. Environmental Health Perspectives 67, 59-74. Tomatis L. (1990)(Editor). Cancer: Causes, Occurrence and Control. p. 201. IARC Scientific Publications No. 100. International Agency for Research on Cancer, Lyon. Watanabe K. and Sato Y. (1971a) Gas chromatographic and mass spectral analyses of heated flavor compounds of beef fats. Agricultural and Biological Chemistry 35, 756-763. Watanabe K. and Sato Y. (1971b) Some alkylsubstituted pyrazines and pyridines in the flavor components of shallow fried beef. Journal of Agricultural and Food Chemistry 19, 1017-1019. Wilett W. C., Stampfer M. J., Colditz G. A., Rosner, B. A. and Speizer F. E. (1990) Relation of meat, fat and fiber intake to the risk of colon cancer in a prospective study among women. New England Journal of Medicine (Dec.), 1664-1672. Yoshida D., Matsumoto T., Okamoto H., Mizusaki S., Kushi A. and Fukuhara Y. (1986) Formation of mutagens by heating foods and model systems. Environmental Health Perspectives 67, 55-58. Yoshida D. and Mizusaki S. (1985) Formation of mutagens by heating amino acids with addition of hydroquinone. Agricultural and Biological Chemistry 49, 1199-1200.
0278-6915/93$6.00+ 0.00 Copyright © 1993PergamonPress Ltd
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I N F L U E N C E OF O X I D I Z E D D E E P - F R Y I N G F A T A N D I R O N ON THE F O R M A T I O N OF F O O D M U T A G E N S IN A M O D E L SYSTEM M. JOHANSSON* and M. J.~GERSTAD Department of Applied Nutrition and Food Chemistry, Chemical Center, University of Lund, PO Box 124, S-221 00 Lund, Sweden (Accepted 15 June 1993)
Abstract--The effects of oxidized fats, iron and tocopherol content on the yield and species of mutagenic heterocyclic amines were studied using a model system. A mixture of glycine (0.9 mmol), creatinine (0.9 mmol) and glucose (0.45 mmol) was heated for 10 and 30 min at 180°C, with the addition of iron and fats. The mutagens formed were identified and quantified using HPLC. (2-Amino-3-methylimidazo[4,5-f]quinoxaline) (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) and 2-amino-3,4,8trimethylimidazo[4,5-f]quinoxaline(4,8-DiMelQx) were formed in the model mixtures. The addition of iron (FeSO4) or oxidized fats to the model system did not affect the species of food mutagens formed, but the iron addition more than doubled the amount of MelQx. The oxidation status of the fat added to the model system had little effect on the formation of MelQx. The fat content was shown to affect the mutagen formation significantly,especiallyafter heating for 30 min. No difference in yield of MelQx was observed in the presence of tocopherol and tocotrienol at naturally occurring concentrations.
(150-250°C) (J/igerstad et aL, 1983 and 1991). The imidazo part of the molecule is assumed to be formed Frying or broiling of meat and fish products at high from creatinine, transformed from creatine by heattemperatures may generate several mutagenic hetero- ing. The imidazo moiety reacts with the quinocyclic aromatic amines, these being either amino- line/quinoxaline part, formed from Maillard reaction imidazoazaarenes or amino acid pyrolysates (Felton products, such as pyridines, pyrazines and Strecker and Hatch, 1986; Sugimura and Sato, 1983). The aldehydes. The suggested precursors behind the foraminoimidazoazaarenes have an imidazo group mation of the aminoimidazoazaarenes have been linked to either a quinoline [e.g. MelQ (2-amino- confirmed in model experiments (J/igerstad et al., 3,4-dimethylimidazo[4,5-f]quinoline)], a quinoxaline 1991). [e.g. MelQx (2-amino-3,8-dimethylimidazo[4,5-f]Lipids have been demonstrated to enhance the quinoxaline)] or a pyridine [e.g. PhlP (2-amino-1- yield of pyrazines, pyridines and Strecker aldehydes methyl-6-phenylimidazo[4,5-b]pyridine)] and are in model systems (Arnoldi et al., 1987 and 1990; suggested to be formed from creatinine, free amino Buttery et aL, 1977; Kawamura, 1983; Parihar et al., acids and monosaccharides naturally occurring in 1981; Watanabe and Sato, 1971a,b). One may thereprotein-rich foods, at normal cooking temperatures fore expect the yield of heterocyclic amines to be increased by lipids. This was also shown in a previous study conducted at our laboratory (Johansson et al., *To whom correspondence should be addressed. 1993) by heating the precursors of the heterocyclic Abbreviations: BHA=butylated hydroxyanisole; BHT= butylated hydroxytoluene; DCM=dichloromethane; amines in a model system containing oils such as corn 4,7-DiMelQx = 2-amino-3,4,7-trimethylimidazo[4,5-f]- oil and olive oil. However, heating the precursors in quinoxaline; 4,8-DiMelQx = 2-amino-3,4,8-trimethyl- the presence of glycerol or various fatty acids had imidazo[4,5-flquinoxaline; 5,8-DiMelQx= 2-amino(except in the case of stearic acid) no effect on the 3,5,8-methylimidazo[4,5-f]quinoxaline;7,8-DiMelQx = 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline; yield. IQ = 2-amino-3-methylimidazo[4,5-f]quinoline; IQx = The possibility that lipids, by being oxidized, might 2-amino-3-methylimidazo[4,5-f]quinoxaline; IS = affect the yield of the heterocyclic amines was not internal standard; iso-IQ=2-amino-l-methylimidazosystematically investigated in our previous study [4,5-f ]quinoline; LC = liquid chromatography; MelQ = (Johansson et al., 1993). Lipid oxidation is known 2-amino-3,4-dimethylimidazo[4,5-f]quinoline;MelQx = to produce free radicals (Eriksson, 1987) and free 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; MS= mass spectrometry; PG = n-propylgallate; PHIP = radical reactions have been proposed to increase 2-amino- l-methyl-6-phenylimidazo[4,5-b]pyridine; the formation of heterocyclic amines (Barnes and SIM =selected ion monitoring; T B H Q = t e r t - b u t y l hydroquinone; 4,7,8-TriMelQx = 2-amino-3,4,7,8-tetra- Weisburger, 1984; Namiki and Hayashi, 1983; methylimidazo[4,5-f]quinoxaline; TLC = thin-layer Yoshida and Mizusaki, 1985; Yoshida et al., 1986). chromatography. Furthermore, lipid hydroperoxides might produce INTRODUCTION
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aldehydes, which could react with amino acids to give Schiff base adducts in analogy to the Maillard reaction (Gardner, 1979). Also, through autoxidation of lipids, carbonyl compounds may be formed that can react with amino compounds to produce browning products of high molecular weight, as reported by Kawamura (1983). The main purpose of this study was to investigate whether the oxidation of lipids affects the yield and principles of heterocyclic amines in a model system. Furthermore, this study was performed to investigate the effect of iron and the combination of oxidized fat and iron on the formation of heterocyclic amines. Both lipids and iron compounds are present during the cooking of meat and fish, in native form (fat, haem iron) as well as in added forms (such as oil, margarine and iron by leaching from the cooking equipment). Iron is known to act as a pro-oxidant in lipid oxidation during the formation of free radicals, which might potentiate the Maillard reaction. On the other hand, unoxidized oil contains tocopherols, which might have a protective effect in their role as free radical scavengers. Thus, if free radicals have any impact on the yield of heterocyclic amines, vitamin E could have a protective role. This was evaluated by comparing the effects of unoxidized oil with those of oxidized oil. Studies of factors behind the formation of heterocyclic amines produced during the cooking of meat and fish are motivated by the fact that such compounds, when tested in rodent bioassays, have been shown to be multipotent carcinogens (for a review see Ohgaki et al., 1991). Dietary fats are also known to increase the risk of cancer, according to several hypotheses that are under investigation (Committee on Diet, Nutrition, and Cancer, 1982; Henderson, 1990; Tomatis, 1990; Wilett el al., 1990). None of them include the possibility that dietary fats might promote the formation of food carcinogens such as the heterocyclic amines, which is an aspect that is investigated in the study described here. MATERIALS AND METHODS
Chemicals. All commercially available chemicals were of analytical grade. Water was taken from a Milli-Q water purification system (Millipore). All solvents [acetonitrile, methanol, dichloromethane (DCM), heptane and di-isopropylether] were purchased from Merck A G (Darmstadt, Germany). [~4C]MelQx [10 mCi/mmol, 98% radiochemical pure according to nuclear magnetic resonance spectral analysis and thin-layer chromatography (TLC)], synthetic MelQx standard solution (100mg/ml, estimated to be greater than 98% pure by TLC and melting point) and iso-IQ (2-amino-l-methylimidazo[4,5-f]quinoline) were purchased from Toronto Research Chemicals (Toronto, Canada). Synthetic IQ (2-amino-3-methylimidazo[4,5-f]quinoline), MelQ,
MelQx, 4,7-DiMeIQx (2-amino-3,4,7-trimethylimidazo[4,5-J]quinoxaline), 4,8-DiMeIQx (2-amino-3,4,8trimethylimidazo[4,5-f]quinoxaline), 5,8-DiMeIQx (2-amino-3,5,8-methylimidazo[4,5-f]quinoxaline), 7,8DiMeIQx (2-amino-3,7,8-trimethylimidazo[4,5-J]quinoxaline) and 4,7,8-TriMeIQx (2-amino-3,4,7,8tetramethylimidazo[4,5-f]quinoxaline) were kind gifts from Professor Kjell Olsson and Dr Spiros Grivas (Department of Chemistry and Molecular Biology, Uppsala, Sweden). IQx (2-amino-3-methylimidazo[4,5-f]quinoxaline) was a kind gift from Mark G. Knize (Lawrence Livermore Laboratory, CA, USA). Ecoscint and Ecoscint A scintillation fluids were obtained from National Diagnostics (Somerville, N J, USA). Dimodan OT (distilled monoglyceride) was obtained from Grindsted (Denmark). Tocopherol standard (~, 7, f-isomer kit, 50 mg per ampoule) was obtained from Merck AG (Darmstadt, Germany). Iron(II)sulfate heptahydrate (FeSO4) was from Merck AG (Darmstadt, Germany). Corn oil was purchased in a local shop. Five partially oxidized samples (nos 1-5) of a deep-frying fat were manufactured by Karlshamns AB (Sweden). The fat consisted of palm oil and hydrated rapeseed oil, which is a common combination in commercially used deep-frying fats. The composition of the fat was as follows: C~4,, (0.6%): Ci6:0 (25.6°/o); Cis:0 (9.1°/oL Cl~:l (54.9%): Ci82 (7.4%); C20:0 (0.6%) and C20:1 (0.6%). A Foodoil Sensor, which measures the dielectric constant of the fat and correlates it to the degree of oxidation, was used to select five partially oxidized samples of the fat (1 =unused fat; 2 =half-used fat; 3 = near upper level for usage; 4 = more oxidized than the upper level for usage according to the Foodoil Sensor, but amounts of polar compounds not exceeding 3 0 ° . which is the recommended upper level for usage according to the Swedish National Food Administration (SLV FS 1990:); 5 = o v e r upper level for usage, exceeding levels of polar compounds and according to the Foodoil Sensor). The samples of oxidized fat were analysed for anisidine value (IUPAC 2.504), peroxide value (AOCS Cd-8-53 modified), free fatty acids, amounts of polar compounds and tocopherol and tocotrienol content (1UPAC 2.432) by Karlshamns AB. The oxidation status of the five partially oxidized fats is shown in Table 1. Model system. The precursors --creatinine (0.9 mmol), glycine (0.9 mmol) and glucose 0.45 mmol)--were dissolved in Milli-Q water, before being heated in closed test-tubes at 180'C for 10 and 30 rain, as described earlier (Johansson et al., 1993). H202 (100#i, 30%) was added and oxygen passed through the mixture for 1 min in order to initiate free radical reactions. The study was performed in three different experiments. In the first, the precursors were dissolved in 2.5 ml Milli-Q water; in the second and third experiments 0.5 g (20%) and 1.0 g (40%) respectively, of one of the five deep-frying fat samples were melted with 5 nag Dimodan OT before addition
Oxidized fats, iron and food mutagens of the precursors dissolved either in 2.0 or 1.5ml Milli-Q water. The solutions were shaken until an emulsion appeared, before heating as above. The experiment was repeated with the addition of FeSO4 (0.09 mmol) in the model system. To ensure that D i m o d a n O T had no effect on mutagen formation, creatinine, glycine and glucose were dissolved in Milli-Q water and heated for 10 rain at 180°C with and without the addition of D i m o d a n OT. Another experiment was performed by heating creatinine, glycine, glucose, corn oil and D i m o d a n O T dissolved in Milli-Q water for 10 min at 180°C without H202 or oxygen. For the determination of vitamin E in the heated mixtures, creatinine, glycine, glucose and D i m o d a n O T were heated for 10 and 30min at 180°C with deep-frying fat number 1 after treatment with H202 and oxygen, as described above. The crude fat (no. 1) was also heated for 10 and 3 0 m i n at 180°C. All experiments were performed in triplicate. Purification. The heated samples were spiked with 8.1 nCi [14C]MelQx serving as an internal standard (IS) before purification. The samples were extracted according to the method of Gross (1990) with some minor modifications (Johansson et al., 1993). After purification, the samples were dissolved in 100/al methanol. Aliquots (50%) of the samples were used for the determination of [14C]MelQx (IS) recovery, using a liquid scintillation counter (1219 Rackbeta LKB Wallac, Sweden). H P L C fractionation. The sample volume was reduced to dryness under nitrogen and finally dissolved in 125 #1 H P L C buffer A (see below). Aliquots (15/al) of the sample were injected into a Varian 9010 Liquid C h r o m a t o g r a p h (LC), with a photo-diode array UV detector (Varian 9065, Polychrom), equipped with an O D S 80 column (ToyoSoda TSK gel TM, 250 x 4.6 mm i.d., 5 ~tm particle size, Varian, Stockholm, Sweden) and eluted with a mobile phase of 0,01 M triethylamine in water adjusted with acetic acid to p H 3.2 (A) and p H 3.6 (B), and acetonitrile (C). Gradients of 5-15% C in A for 10min, then 15-25% C in B for 10rain, and finally 25-55% C in B for 5 min were used. The flow rate was 1.0 ml/min and the effluent was monitored at 263 nm. In some experiments, fractions were collected every 30 sec
973
between 8 and 30 min and diluted before the assay of mutagenic activity. Mutation assay. The mutagenic activity of the fractions was tested as described by Ames et al. (1975) using Salmonella strain T A 98 with the addition of 0.5 ml S-9 mix containing 5% chlorophene-induced rat liver per plate (Maron and Ames, 1983). The fractions were tested at one dose (25 pl out of a total volume of 1 ml). Synthetic M e l Q x was used as a positive control (50,000 revertants//zg). The colonies were counted in an automated colony counter (Bio Tran II, New Brunswick Scientific Co, Inc., N J, USA). The spontaneous reversion rate was 30-35 revertants/plate. A fraction was considered mutagenic if it induced a number of revertants that was twice the background level. H P L C identification and quantification. The mutagens produced in the model system were fractionated using HPLC, as described above. The identities of the mutagens were established by comparing the retention times of the mutagenic peaks, according to the Ames test, with the retention times of synthetic compounds, namely IQ, iso-IQ, IQx, MelQ, MelQx, 4,7-DiMelQx, 4,8-DiMelQx, 5,8-DiMelQx, 7,8D i M e l Q x and 4,7,8-TriMelQx, obtained under the same conditions. In addition, some samples were also spiked with synthetic compounds before injection. Diode array UV spectra of the peaks were compared with diode array UV spectra of synthetic compounds obtained under the same conditions. The mutagens were quantified by comparing the peak area of the H P L C chromatographed sample with the area of a known amount of standard. The amount was corrected for the recovery of [a4C]MelQx. M S identification. Two of the mutagens formed in the model system were identified earlier as MeIQx and 4,8-DiMeIQx using mass spectrometry (MS) (Johansson et al., 1993). The third mutagenic fraction isolated from a heated model mixture (creatinine, glycine and glucose heated in 2.5 ml Milli-Q water for 10min at 180°C) was identified using L C - M S with selected ion monitoring (SIM). The mass spectrum obtained was compared with that of synthetic IQx. Determination o f vitamin E. The tocopherol and tocotrienol content (~-, 7- and 6-forms) were determined in the crude fat (no. I), heated crude fat (no. 1)
Table 1. Foodoil Sensor*, peroxide value, free fatty acids, anisidine value, polar compounds, tocopherol and tocotrienol contents in the partially oxidized deep-frying fats (nos I-5) (single determinations) Peroxide Polar Tocopherol (T)/tocotrienol (T3) content (mg/kg) Deep-frying Foodoil value Free fatty Anisidine compound fatt Sensor (mEq/kg) acids (%) value (%) -T#t -T 3 y -T/3' -T3 ~-T/6-T 3 1 re~rence 3.2 0.03 4.0 4.4 160/70 180/90 10/20 2 1.6 6.1 0.2 65.9 13.0 <1 <1 <1 3 3.7 5.2 0.4 87.8 25.1 <1 <1 <1 4 5.2 4.5 0.7 94.9 27.8 <1 <1 <1 5 >6 3,2 1.0 97.9 32.6 <1 <1 <1 *The Foodoil Sensor measures the dielectric constant of the fat and correlates it to the degree oif oxidation. tl = unused fat; 2 = half-used fat; 3 = near upper levelfor usage; 4 = more oxidized than the upper level for usage according to the Foodoil Sensor, but amounts of polar compounds not exceeding30% (the recommended upper levelfor usage according to the Swedish National Food Administration).
974
M. JOHANS.,tK)Nand M. JJ~GERSTAD
E t-
@ ¢q O O
O
[q 8
400 200 0
1
5
10
15
20
25
min
Fig. 1. UV absorbance at 263 nm and mutagenic activity in (revertants/fraction) in fractions collected from HPLC fractionation of a mixture of creatinine, glycineand glucose heated at ! 80°C for 10 min. Peaks at retention times 11, 15 and 18min correspond to 2-amino-3-methylimidazo[4,5-f]quinoline(IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMelQx), respectively.
and in model systems containing fat (no. 1) and precursors, heated for 10 and 30 min at 180°C. The vitamin E forms were quantified using an HPLC (Varian 5000 Liquid Chromatograph) with a fluorescence detector (Shimadzu Spectrofluorophotometer, RF-540) set at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. The HPLC was equipped with a Lichrosorb Si-60-5 column (Chrompack 250 x 4.6 mm i.d.), and eluted isocratically with a mobile phase of heptane:diisopropylether (91:9), at a flow rate of 2.5 ml/min (H~kansson et al., 1987). 20/~1 of the samples (only the fat phase was taken from the heated model mixtures) dissolved in heptane (200mg sample/ml heptane for the crude samples, 400 mg sample/ml heptane for the model mixtures) and standard solutions (ct-, y- and 6-tocopherols, 10#g/ml heptane) were injected onto the HPLC column. Statistical evaluation. The differences between the samples ( n > 12) were evaluated statistically with Student's t-test using a PC version of Minitab.
RESULTS Mutagens produced in the model system Three major mutagenic compounds were formed in the heated model mixtures, according to the results of the Ames test on fractions from HPLC. The same mutagenic pattern was observed for all heated mixtures containing any of the fats and regardless of whether FeSO4 was added or not. In Fig. 1 the mutagenic activity from a heated mixture and a corresponding chromatogram are shown. Two of the mutagenic compounds were earlier identified as MelQx and 4,8-DiMelQx using MS. In the present study, their identities were confirmed by comparing their UV spectra with the UV spectra of synthetic references. The third mutagenic fraction corresponded to synthetic IQx in retention time and UV spectrum. Furthermore, the peak co-eluted with synthetic IQx when fractionated on HPLC. The presence of IQx was confirmed using LC-MS with SIM. In Fig. 2 an LC-MS chromatogram of the fraction containing IQx and synthetic IQx is shown.
975
Oxidized fats, iron and food mutagens
Effects of oxidized fats and iron on the formation of MelQx The quantitative results from the HPLC analysis of the heated mixtures are shown in Fig. 3. The amounts were corrected for [14C]MeIQx recovery, which varied between 52 and 97%.
Heating the precursors (creatinine, glycine, glucose, H202 and oxygen) in the absence of fats at 180°C for 10 and 30 min produced 3.4 and 10.4 nmol MeIQx/mmol creatinine, respectively, if no FeSO4 was present and 10.6 and 17.2nmol MeIQx/mmol creatinine, respectively, in the presence of FeSO4. The amount of MeIQx formed was significantly increased
11:57 100
50
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.
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.
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.
.
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.
6:40
.
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i
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.
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.
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.
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.
.
.
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13:20
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,~
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12:00 I00
50
3:20
6:40
10:00
13:20
Fig. 2. Plasma-spray mass spectra. Single-ion recording of the protonated molecular ion (MH+). Top: mutagenic fraction from a heated (180°C/10 min) mixture of creatinine, glycine and glucose. Bottom: synthetic IQx.
mL~
976
M . JOHANSSON a n d M . J~GERSTAD
No fat 17,- - "1 . . . . . . . . . Fat no 1 20% I . . . . . . Fat n o 2 2 0 %
I...~
] ....... ..........
Fat n o 3 20% r. 7 ~ T . '
.....
~--.
Fat no 4 20% k ¢ ¢ ~ f i t . . . . . Fat no 5 20% 7 Fat no 1 4 0 %
7-7'' 1 .......
Fat no 2 4 0 %
¢ ....
Fat n o 3 4 0 % I . . . . . Fat no 4 4 0 % Fat n o 5 4 0 %
_
. . . . . . . . . . . . . . ~. . . . . . . .
--
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............. ~ I 0
....... I 5
I 10
I 15
I 20
I 25
30
nmol MelOx/mmol creatnine Fig. 3. Amounts of MelQx (nmol MelQx/mmol creatinine) formed in model systems containing creatinine, glycine, glucose, deep-frying fat (nos 1 5), Dimodan OT, H202 and oxygen, with and without the addition of FeSO4. All values are means of three measurements: 71, 10 min at 180°C without iron; [], 10min at 180°C with iron; D, 30 min at 180°C without iron; II, 30min at 180°C with iron. (P < 0.001) when the model mixture was heated at 180°C for 30min compared with 10min, both with and without iron and any of the fat samples. The degree of oxidation of the fat had small effects on the formation of MelQx in the model system. As can be seen in Fig. 3, no consistent trend was demonstrated between the yield of MelQx and the degree of oxidation. When comparing all mixtures containing any of the fats, significantly higher (P <0.05) amounts of MelQx were formed in model mixtures containing 40% fat than with 20% fat. Comparing the MelQx content in model mixtures without fat, and mixtures with 20% or 40% of all fat samples, showed significant differences (P < 0.05) only when an iron-containing model mixture was heated for 30 min with 20% fat or when a mixture with or without iron was heated for 30 min with 40% fat. The addition of iron (FeSO4) significantly increased the formation of MelQx more than two-fold (P < 0.001), when the mixture was heated for both 10 and 30min at 180°C. Similar amounts of MelQx were formed both in a mixture containing Dimodan OT, heated for 10 min at 180°C, and in a mixture containing no emulsifier, 6.7 and 4.0 nmol MelQx/mmol creatinine, respectively. Thus, neither the addition of a monoglyceride as an emulsifier nor the addition of H202 and oxygen in the model mixtures had any quantitative effect on mutagen formation (P > 0.05). V i t a m i n E content
Only sample no. 1 contained natural tocopherols and tocotrienols, whereas all vitamin E activity had been destroyed in the more oxidized samples (nos 2-5) (Table !). The tocopherol and tocotrienol content in crude unheated fat (no. 1), heated fat (no. 1) and in a heated model system containing fat (no. 1) is shown in
Table 2. As can be seen, the ~-tocopherol and 7tocotrienol contents were affected most, but were similar in the model mixtures and the heated fat. The 7-tocopherol and ),-tocotrienol contents were lower when the fat or model system was heated for 30 min than when it was heated for 10 min. The 6-tocopherol and 6-tocotrienol contents were not affected by heating in any of the cases, being 10 and 20 mg/kg for 6-tocopherol and 6-tocotrienol, respectively. DISCUSSION
Three major mutagenic peaks were identified in all the heated mixtures. Thus, the addition of fat, oxidized fat or iron did not affect the principles of the heterocyclic amines. This has been shown previously (Johansson et al., 1993) with the addition of different fatty acids and oils in a similar model system. The mutagenic peaks corresponded to IQx, MeIQx and 4,8-DiMelQx, respectively. The formation of MelQx, 4,8-DiMelQx and also 7,8-DiMelQx from creatine, glycine and glucose in model systems in the absence of fat, has been reported earlier (Jiigerstad et al.,
Table 2. Tocopherol (T) and tocotrienol (T 3) content (mg/kg) in unheated crude fat (no. 1)*, heated fat (no. 1) and in heated model mixtures containing fat (no. 1) (means of two determinations) ~-T/0:-T 3
7-T/y-T 3
6 -TI6-T 3
Unheated fat
190/70
240/I 10
10/20
Heated fat (180 C / 1 0 min)
110/30
190/80
10/20
Heated fat (180 C/30 min)
40/10
110/50
10/10
Model system (180~'C/10 min)
110/30
170/80
10/20
Model system (180°C/30 min)
80/20
150/70
10/20
Sample
*As in Table 1.
Oxidized fats, iron and food mutagens 1986 and 1991; Negishi et al., 1984; Skog and J/igerstad, 1990). The formation of IQx has only previously been reported in a dry-heating experiment involving serine and creatinine (Knize et al., 1988). However, IQx might have been formed before, but escaped detection by being lost during the commonly used XAD-2 purification (Felton and Hatch, 1986). Both MeIQx and 4,8-DiMeIQx are usually formed in fried beef (Felton and Knize, 1990). IQx has only been found in a fried meat emulsion with added creatinine (Becher et al., 1988). The yield of MeIQx was shown to increase significantly with increased heating time (10-30 min), lipid concentration (20--40%) and by adding FeSO, (the Fenton reaction). The addition of an emulsifier, Dimodan OT, did not affect the yield, neither did the presence of hydrogen peroxide and oxygen. The results support those of our previous study (Johansson et al., 1993) also showing increases in the yield of MeIQx in the presence of oil and with increased heating time. The observation that deep-frying oil affected the yield of MeIQx is in agreement with previous studies demonstrating an enhancing effect of lipids on the formation of Maillard reaction products, e.g. pyrazines, pyridines and Strecker aldehydes (Arnoldi et al., 1987 and 1990; Buttery el al., 1977; Kawamura, 1983; Parihar et al., 1981; Watanabe and Sato, 1971a,b). As these products are assumed to be precursors of the IQ compounds (J/igerstad el al., 1983 and 1991), an increase in their formation should be associated with a corresponding increase in the yield of IQ compounds. It was also of interest to investigate whether oxidized lipids had any specific effect on the yield of the heterocyclic amines. As each oxidized oil sample tested was investigated only in triplicate, it was not possible to evaluate the results statistically. However, although there were some differences in mutagen formation between the fat samples, there seemed not to be any consistent trend with the degree of oxidation. This might perhaps be attributable to the addition of hydrogen peroxide and oxygen to all samples regardless of degree of oxidation, thereby possibly masking differences in their effects on the yield of MeIQx. However, in a separate experiment, we found no effect on the yield of MeIQx by adding oxygen and hydrogen peroxide to the model system. The reason why all the samples were treated with hydrogen peroxide and oxygen was to create more favourable conditions for the oxidized oils to produce free radicals during heating in the model system. This experimental design does not allow any conclusions to be drawn on the effects of oxidized fat p e r se on the formation of MeIQx. However, the results seem to indicate that free radicals produced from the fat oxidation catalysed by H202 and oxygen did not have any marked influence on the yield of MeIQx. Interestingly, the Fenton reaction (Fe :+ + H:O2--*Fe3+ + O H - + O H ' ) doubled the yield of MeIQx (P < 0.001), both when the mixtures were
977
heated for 10 and for 30min at 180°C. The iron catalyses extremely reactive free radicals, such as lipidperoxy radicals. The doubling of the MeIQx yield by the Fenton reaction was seen both in the presence and absence of fats. Iron has previously been reported to enhance the formation of food mutagens in fried beefburgers. Barnes and Weisburger (1984) added 10 ppm Fe 2+ and Fe 3+ to meat before frying and reported a doubling in mutagenic activity. The authors suggested that the catalytic effect of iron ions was related to their capacity to accelerate free radical reactions. Taylor et al. (1986) reported that FeSO4 increased the formation of IQlike compounds by nearly doubling the mutagenic activity in a model mixture consisting of creatine phosphate and tryptophan. Moreover, the findings reported by Parihar et al. (1981) indicate that iron and copper ions catalyse the formation of pyrazines and pyridines when heating oxidized oil and amino acids in the absence of sugar. Thus, the mechanism by which free radicals might affect the yield of MeIQx is probably through an increase of precursors such as pyrazines and pyridines, although other mechanisms such as activation of the reactants cannot be excluded. The enhancing effect of iron is very interesting since there are two possible iron sources during the frying of meat--haem-iron released through the denaturation of myoglobin present in animal muscle, and by leaching from the cooking equipment itself. The haem-iron source is difficult to influence, but iron leaching from cooking equipment might be reduced by using iron-free material such as teflon [poly(tetrafluoroethene)]. The enhancing effect of free radical reactions on the yield of MeIQx seems to be supported by the Fenton reaction, but the results of this study indicate that lipid oxidation without catalysts is insufficient to induce enough free radicals to affect the yield of MeIQx. Vitamin E is a natural lipid antioxidant known to be an effective free radical scavenger (Burton and Ingold, 1981). The least oxidized fat sample (no. 1) was the only sample that contained vitamin E (640 ppm); however, this sample produced a yield of MelQx similar to those in samples lacking vitamin E. During the heating of oil in the model system, vitamin E was partly lost, but between 20 and 30% of the ~t-forms resisted heating and more than half of the ~and c~-forms were present after 30 min heating. Thus, the presence of vitamin E in sample no. 1 seemed not to have any pronounced inhibitory effect on the yield of MeIQx, regardless of whether iron was present or not. If free radicals are an important contributory mechanism for the formation of heterocyclic amines, the sample containing natural vitamin E would have exhibited a lower yield of MeIQx than the other oxidized deep-frying oils, but this was not the case. Perhaps the concentration of vitamin E needs to be considerably higher in order to exert inhibitory effects. Studies have been performed by Pearson et al.
978
M. JOHANSSONand M. J~GERSTAD
(1992) and Chen et al. (1992) on the effect of different natural and synthetic antioxidants during the frying of beef. Vitamin C (100ppm), vitamin E, B H A (butylated hydroxyanisole), P G (n-propylgallate) and T B H Q (tert-butyl-hydroquinone) (0.1% of fat content) decreased the formation of IQ compounds by about 50-70%. In contrast BHT (butylated hydroxytoluene) (0.1% of fat content) enhanced the formation of 4,8-DiMelQx by 12%. The amounts of the other two heterocyclic amines identified, 4,8-DiMelQx and IQx, were roughly estimated to be 1-2 and 9 nmol/mmol creatinine, respectively. Neither of these two compounds seemed to be affected by heating time, lipid concentration, degree of oxidation or by the Fenton reaction. However, since the quantification of these two compounds was not based on recovery corrections performed by spiking the samples, it is not possible to draw any definite conclusions about these two IQ compounds regarding yield-affecting factors such as lipids and free radicals. In conclusion, the study described here demonstrates a significant chemical effect of lipids on the yield of MelQx, catalysed by iron and probably mediated by the Maillard reaction. These findings add further yield-affecting factors to the formation of the heterocyclic amines. In a previous study, we have shown that lipids affected the amount of mutagenic activity by creating more efficient heat transfer during the cooking of meat loaf (Holtz et al., 1985). Thus, one explanation of the increased formation of heterocyclic amines during the cooking of products high in fat or with added fat could be simply a temperature rise. The model system used here excluded such a possibility, since the heating was performed under strictly controlled conditions. It therefore seems that lipids enhance the formation of IQ compounds both by physical and chemical means. Previous studies reported in the literature have shown lipids to have enhancing effects, no effect or even inhibitory effects (Barrington et al., 1990; Bjeldanes et al., 1983; Knize et al., 1985; Nilsson et al., 1986; Spingarn et al., 1981). In general, these studies do not discuss the yield-affecting mechanisms; furthermore, most of them rely on measurements of mutagenic activity only, and it is not always clear whether the investigators have excluded possible inhibitory effects of certain fatty acids on the Ames test. An aspect that has hitherto escaped attention is whether naturally occurring antioxidants in meat or oils, such as tocopherols or fl-carotene, might inhibit the formation of heterocyclic amines during cooking. This study did not give support the theory that vitamin E at concentrations occurring naturally in deep-fat frying oil has any protective role in this respect, but further research on this topic is necessary. Acknowledgements--We are grateful to Karlshamns AB, Sweden, for manufacturing and analysing the oxidized fat samples, Drs G. A. Gross and L. Fay, Nestl6 Research
Centre (Vers-Chez-les-Blanc, Switzerland) for performing the LC-MS analysis and Mrs Hanna Nilsson and Gunnel Andersson, Department of Applied Nutrition and Food Chemistry, for their excellent technical assistance. The kind gifts of synthetic references from Professor Kjell Olsson and Dr Spiros Grivas (Uppsala, Sweden) and Mark G. Knize (Livermore, CA, USA) are gratefully acknowledged. Salmonella typhinurium strain TA 98, was kindly provided by Professor Bruce Ames, University of Berkeley (Berkeley, CA, USA). This study was supported by the Swedish Cancer Foundation (1824-B92-11XAC) and the Swedish Council for Forestry and Agricultural Research (50.0440/91). REFERENCES
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