Dietary exposure to heterocyclic amines in high-temperature cooked meat and fish in Malaysia
Food Additives and Contam inan ts
Vol. 27 , No.8 , August 201 0, 1060- 1071
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Taylor &Francis
Taylor£..Fra nclsGroup
Dietary exposure to heterocyclic amines in high-temperature cpoked meat and fish in Malaysia
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M.H.A. Jahurul, S. Jinap*, SJ. Ang, A. Abdul-Hamid, P . Hajeb. H.N. Lioe t and I.S.M. Zaidul
Cenl er of" Excellence /i)r Food S a/i' ly Research (C EFS R ) . Facultv
43400 U PM, S elango r, Malavsia
0/ Foo d Scien ce and Technology, Universili Putra Malaysia.
(R ecci l'cd ]5 janllan ' 20 11!. /il1a l I'crsiun received 2] M arch 20 10 )
The inta ke of hcterocycl ic amin es is influenced by th e am ount and type of mca t and fish inges ted, frequen cy of
consumpti o n, cooking method s. cooking tem pe rature, and du rati on of cooking. In thi s study, the dietary intake
o f heterocyclic amincs in Ma lays ia and th eir main sources were inves ti gated . Fo rt y-two samples of meat and fi sh
were anal ysed by hi gh-perfo rm a nce liquid chromatograp hy with pho todi ode array detector to determin e
the concent rati on of th e six predomin a nt heterocyclic amin cs, namely: 2-amino-3-meth ylimidazo[4,5f]q uin ol ine (I Q ). 2-a mi no-3 A-dimet hylimidazo[4. 5-f lq uino Ii nee 1\1 eIQ). 2-amino- 3,S-dimethylimidazo[4 ,5･ｴ ｨ ｹ ｬｩｭ､
｡ｺｯ
｛ ＴＬＵＭ
ｦ＠ Jquinoxalin e (4,8- DiMeIQx), 2-amino-3 ,7,Sflq uin ox alin c (M e l Qx) . 2-amin o- Ｓ ａ ＬｾＭ ｴｲｩｭ
trim eth yl imida w [4. 5-1' Jq uin oxalin e (7 .S-Di Me l Qx). ,1 nd 2·am ino - I -mcth yl-6-phcnylimidazo[4,5-b]pyridinc
(PhIP). Dietary intak e dat a we re obtained lI sing a food-freq uen cy qu es ti o nn a ire wh en interviewing 600
Ma la ys ia n res pondents. The level of tota l heterocyclIc ami nes in food sam ples studies ranged fr om not detected
to 38.7 ng g- I. T he a ve ra ge dail y intake level of hete ro cycl ic am ine was 553.7 ng per capita day- I. The intake of
PhIP was th e hig hest. followed by MeI Qx and Ivl eIQ. T he result s revea l tha t fri ed a nd grill ed chicken were the
maj or di eta ry so urce o f hetewcycl ic am ines ill ivlalaysia . However. the heterocycl ic a mine intake by the'
Mala ys ian popu lati on was lo \\'c r tha n th ose repor ted fr olll other reg ions
Keywords: exposllt'c a,;sesslll cnt: heterocycli c am in es: co oked fo od,;
Introduction
Heterocycli c a mines (HCAs) a re possible human
a nd animal carcin oge n s fo nned in cooked m eat an d
fi sh during hi g h-temp e ra ture c ookin g processes
(Internation a l A ge ncy for R esea rc h o n Cance r
(IARC) 1993). Th e rAR C a lso reported that
HCAs such a s Ｒ Ｍ ｾ［ ｭｩｮｯ
ＭＳＬ
ＴＭ､ｩｬＱ･ｴｨ
ｹ ｬｩ､｡
ｺｯ ｬＴ Ｎ ＵＭ
fJquinoline,. (MelQ), 2-amino-3,8-dime th ylimd azo [4,5fJquinoxalin e (MeIQx), and 2-a mino-l-meth yl-6-phen ylimidazo [4, 5-b) pyridine (PhIP) are possible huma n
carcinogens;
and
2-am ino-3-me th ylimida zo[4,5-f]quinoline (IQ) a s a probabl e ca rcinogen. H C A s a lso
ca use cancer at a varie ty of multiple sites in labo ratory
a nimal species (EI-Ba y oum y et al. 1995), Several ca secontrol studies in various p o pula tion s h ave repo rted
positive associati o n s between a highe r intake of wellcoo ked meat and th e risk of colon cancer (Sinh a e t al.
1999), breast ca ncer (Sinha e t a l. 2000), lun g ca nce r
(Sinha 1998), and gas tric cance r (de Stefani et a l. 1998).
Huma n s arc ex p o sed to p o te nti a ll y to x ic s u bstances
including natura lly occ urrin g muta geni c and carci n o ge nic c o mpoun ds . On e's daily di e t lll ay c o nt ai n H C A s
a nd polycyclic a romatic hydroca rbon s ( PAH s) as a
res ult of high-temperature processi ng o f m eat a nd fi sh .
Bo t h gro ups a re eas il y found in grilled and fri ed meat,
p o ultry an d fi sh (Gomaa et al. 199 3; Kaze rouni e t al.
200 I; J iigersta d a nd Skog 20(5), T he present study is
foc llsed on H C A s. Friedman (1996) reported that some
H CA s pl ay a n im p o rtant rol e in th e taste, fla vour, a nd
eve n smell o f h ea ted meat. H C A s are form ed by th e
pyro lys is of pro te in a nd amino acids in pro tein-rich
food s. Th e ir form a tion depend s on several factor s such
a s tempe rature, c ooking method and time, the degree
of d o ne ness, th e c oncentration of HCA prec ursors, and
th e presence of co mpounds with enhancing or inhibit o ry e ffects (Felto n et al. 2000; Busquets et a L 2004) .
There has bee n a large variability of estimated intake
of di eta ry H C A s reported from different countries
(suc h as Japan , Singapore , Sweden , Switzerland ,
Spa in , th e Unite d Kingdom, a nd the United States).
The rea so n ma y be due to diffe rent cooking method s,
cook in g te mperature. duration of cooking, different
ques tio nnaire m a ke-up, and th e studied populations .
ｈ ｯ｜Ｇ
･ ｜｣ｾ ｲＬ＠ fo r th e in ves tigation o f hum a n ca rcinogen s,
an a cc ur a te assess m e nt of th e indi vidua l co nsumption
of H eAs is esse nt ia l. U p-to-dat e . exte nsive research on
HCA s has been p e rfo rm ed on th e W es tern die t
(Wakabay a shi et a l. 1992; S kog 199 3; Kni ze et al.
1995: Z il11ll1c rli et a l. 2001: Sk og a nd Sol ya kov 2002 ;
Ｊｃ ｯ ｲ ･ｳｾ
ｮ､ｩ ｧ＠
author. Emails:jinap@ food.upm.edu .my:sjinap@gmail.co m
' Present address : Depa rtm ent of Food Science an d Tec hn ology, Bogor Agri cultura l University, Bogo r, Indonesia
ISSN 0265-203X pri nt/lSS N 1464-51 22 o nl ine
ro 10 10 Tayl or & Francis
DO l: tO I080i]944004 1003801 190
http: // ww w.in fo rillaw orld .com
Fo od Additives and Contaminants
Cantwell et ai. 2004; Llobet et a!. 2006: Domingo et a!.
2007; Marti-Cid et ai. 2007). Fried meat is an important source of exposure to HCAs in the Western diet.
Although HCAs are formed at ppb levels (ngg- I),
their intake may be dangerous for human health
because of the total amount of cooked meat or fish
consumed (Busquets et a1. 2004).
This is the first extensive study on HCA exposure in
Malaysia. The analysis of Malaysian foods for the
occurrence of HCAs is important because there is
worldwide human exposure to these compounds
according to many reports (Augustsson et a!. 1997:
Byrne et ai. 1998; Kobayashi et ai. 2002; Wong et ai.
2005). The aim of the present study was to determine
the level of major six HCAs - IQ, MeIQ, MelQx, 4, 8DiMelQx, 7, 8-DiMeIQx and PhIP - in some of the
most commonly consumed foods in Malaysia in order
to estimate the exposure of the Malaysian population
to these toxic compounds. The assessment was performed by combining the content of HCAs in cooked
food samples analysed in the laboratory with information on food consumption from the food frequency
questionnaire.
Materials and methods
Study population and questionnaire
A cross-sectional survey was conducted in the central
zone of Peninsular Malaysia covering three locations:
Kuala Lumpur, Putra Jaya, and Selangor state. The
respondents comprised three main races: Malay
(59.5%), Chinese (29.8%), . and Indian (10.2%) , and
other ethl/ie (0.5%), which were almost similar in
proportion to the Malaysian population. A total
sample size of 600 adult respondents aged IS years
and above was selected randomly for the survey. One
adult was chosen to represent the pattern of the whole
household. The sample size was measured by using a
two-staged stratified random sampling method and the
following equation was applied in the sample size
estimation:
ｎ
-Ｍｾ
1.96 2 x P(I - P)
de
(I)
where a Z-value of 1.96 for p=0.05 (95% confidence
level); P is the estimated prevalence of the sample equal
to 0.5; and d is a margin of error of 0.1 for ± I 0%.
In the first stage of stratified random sampling, 75
enumeration blocks were selected from living quarters
where each living quarter consists of between 80 and
120 houses. In the second stage of stratified random
sampling, eight households were selected from each
enumeration block. The enumeration blocks and living
quarters for the survey were provided by the
Department of Stati stics Malaysia .
1061
Study design and questiollnaire
The questionnaire was divided into three sections:
cooking utensils and cooking methods used at home;
demographic data of the respondent; and dietary
intake. Dietary intake was assessed using a food
frequency questionnaire with a 1-month recall. The
food frequency questionnaire included information on
food items, the frequency of food consumption and the
portion size of food ingested. Before the main survey,
a pilot survey with 60 respondents was performed in
the targeted areas to test question planning in the
questionnaire.
Selection o/food items and samplillg offoods
Food item s were selected based on the literature
publi shed in peer-revievied journals, which included
all types of piocessed (such as drying and smoking)
and cooked (such as grilling, roasting and frying) foods
report ed to contain !-leAs . There were in total 42 types
of food item s; three samples were purchased from the
same stall at different times of the day. The selected
meat and fish dishes for this study were fried fish, fried
chicken (deep-fried), chicken soup , chicken curry,
steamed fi sh. chi cke n cooked with chilli (masak
merah). chicken burger, {ish curry, fried chicken,
keropok kkor, chicken satay. chicken randang, fish
fried in chilli, nu gge t, fish in tamarind (asam pedas
Ikan), paprika chicken, sweet-and-sour fish, sausage,
fish soup , chicken in coconut milk gravy (ayam masak
lemak), fri ed salted fish, fish in coconut milk, chicken
percik (ayalll percik), fried beef, beef cooked with
chilli, roasted fish, beef curry, smoke fish, fish in soya
sa uce , beef burger, beef satay, beef rendang, chicken
golek (ayam golek), fish in tauClI sauce, beef soup,
chicken kurma, mutton satay, paprika beef, fish with
three navours , grilled chicken tandoori, mutton curry,
beef in coconut milk gravy (masak lemak daging),
barbecue-grilled chicken, black pepper-grilled chicken,
beef kurilla , and honey-grilled chicken.
Samples of all food items were purchased from
local food stalls in Kuala Lumpur, Selangor, and
Putrajaya. Upon arrival in the laboratory, samples'
bones were removed from the meat and fish , then
samples were homogenized using a Waring blender
(Dawsonville, GA , USA). The homogenized samples
were then pooled and mixed together before being
quarter sampled. The representative samples were
stored at -18 v C until analysis.
Statistical analysis
Statistical analyses were performed using SPSS
(version 14.0). The reliability analysis was measures
as an internal consistency. Cronbach's alpha was the
measurement for internal consistency. The survey was
1062
Af.H.A. Jahllrlll et aJ.
considered to be reliable if Cronbach's alpha was at
least 0.7. The descriptive analysis, including the mean,
median, mode, standard deviation, and frequency of
food taken by respondents, was used to describe the
basic features of the data collected from the survey. An
independent sample {-test was used to compare the
differences between two genders. Analysis of variance
(ANOVA) was used to test the differences between
different HCAs in different food items. A p < 0.05 was
considered to be statistically significant.
Chemicals
Solvent and chemicals were of both high-performance
liquid chromatography (HPLC) and analytical grade.
The water was purified in an Elix-MilliQ system
(Millipore, Bedford, MA, USA). All solutions were
passed through a 0.45-l1m filter before injection into
the HPLC system. HCAs standards, i.e . 2-amino-3methylimidazo[4,5-f]quinoline
(lQ),
2-amino-3,4dimethylimidazo[4,5-f]quinoline (MelQ), 2-amino3 ,8-dimeth y Iimidazo[ 4, 5-f]q uinoxaline
(Mel Qx),
2-ami no- 3,4,8- trimethylimidazo[ 4,5-f]quinoxaline (4,8DiMeIQx),
2-amino-I-methyl-6-phenylimidazo[4,5b]pyridine
(Ph I P),
2-amino- 3, 7 ,8-trimethylimidazo[4,5-f]qunoxaline (7,8-DiMeIQx), and 2-a111ino3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline
(4,7,8TriMelQx) as internal standard, were purchased from
Toronto Research Chemicals (Dowl1sview. ON,
Canada). Empty Extrelut-20 extraction cartridges
were provided by Merck (Darmstadt, Germany) and
Oasis MCX cartridges (3 mli60 mg) by Waters
(Milford , MA, USA). MCX cartridges were preconditioned with ethyl acetate (2 ml). The solute
diatomaceo,us earth refill material was obtained from
1ST (Hengoed, UK).
RCA extraction
The extraction and clean-up of all six HCAs studied
was carried out following the modified method of
Gross and Griiter (1992) and Messner and Murkovic
(2004). Samples (I g) spiked with internal standard
(4,7,8-TriMcIQx, 250 ppb) were dissolved in 12ml of
1 M NaOH and the suspension was homogenized using
magnetic stirring for 3 h with 500 rpm (Heidolph, MR
3001 K). The alkaline solution was mixed with diatomaceolls earth (13 g) , and then transferred into empty
Extrelut columas. Ethyl acetate was used as the
extraction solvent and elute was passed through the
coupled Oasis MCX cartridges. The MCX cartridges
were washed with 0.1 M HCI (2 ml) and MeOH
(2 ml). The analytes were eluted with 2 ml of
MeOH-concentrated ammonia (19/1, vjv). The samples were evaporated to dryness under a stream of
nitrogen and the final extracts were dissolved in 100 pi
methanol just before analysis for HCAs. Recovery of
HCAs was determined using the standards addition
method. The standards mixture (200 and 500 ng g- I
sample) was spiked to the chicken, beef, and fish
samples before the extraction step. HCAs were separated using reversed-phase LC and a TSK gel ODS T M
column (25 em x 4.6 mm. 5 11m). The LC systems consisted of a Waters LC model 2690 (CA, USA) with a
photo-diode array detector (Waters model 2996),
equipped with a quaternary solvent delivery system
under a Waters Controller model 600, an autosampl er
(Waters model 717) , and an on-line vacuum degasser.
The mobile phase consisted of solvent A, 0.0 I M
triethylamine in water (adjusted to pH 3.3 with acetic
acid), and solvent B, acetonitrile (HPLC grade;
Merck). Analyses were carried out in a !!radient
mode for 33 min. Gradient programming \;as performed as follows: at zero to 18 min it was 5% to 23%
B in A, at 18- 25 min it was 23% B in A, and at
25--33 min it was 23% to 25% B in A. The flow rate of
the mobile phase was I ml min - I.
Determination of LOD and LOQ
The limit of detection (LOD) and limit of quantification (LOQ) were obtained by performing seven determinations of the lowest acceptable standard
concentration. LOD was calculated as three times the
standard of deviation (SD); LOQ was ten times the SD
of the average background signal.
Estimated dai{.v intake
The estimated daily intake levels of HCAs by an adult
(ng day - I per capita) were calculated using the average
of each meat and fish product, i.e, (Hajeb et a!. 2009):
Daily intake per person per day]
= mean concentration (ngg- I) of HCAs x amount of
RccIJl'er), test
RecO\ery assays were established by spiking known
amounts of the matrices (200 and 500 ng g - I sample) of
the HCA standard solution to samples. Seven replicates
were conducted on each fortified sample. The recovery
assays were calculated by comparing the concentration
of the fortified samples with those of the unfortified
samples.
meat and fish products consumed per day (g per capita)
Results 'and discussion
The socio-demographic characteristics of the study
population are summarized in Table I. The distribution for the gender and the ethnic group of the
respondents represented the Malaysian population.
Food Additives and Contaminants
Table I. Socio-demographic chara cteri stics of respondents.
Tabl e 1. Continued .
Demographic profil e
Demographic profil e
Frequency
%
Gender
Male
Female
303
297
50.5
49.5
Elhniciry
Malay
Chinese
Indian
Othe rs
357
179
61
3
62.5
29.8
10.2
0.5
18- 24
25-29
30-34
35- 39
40--44
45--49
50- 54
55- 59
6064
130
91
64
64
67
52
48
30
22.2
15.2
10.7
10.7
11.2
8.7
65- 69
20
5
3.0"
3.3
ｾＷＰ＠
II
1.8
1063
Frequency
%
394
248
204
200
249
255
65.7
41.3
34
33.3
41.5
42.5
Cooking merhod
Stir-fried
Deep-fried
Boiling
Baking
Grilled
Roasted
Age group (rears )
23
Educa rion /('1'1'/
Uneducated
Primar y sc hoo l
Seco nd ary scho ol
College
Bachelor deg ree
Postgrad uCJt e
Others
Carccr categories
Government se r va nt
Pri va te sec to r
Self-employed
Farmer
Student
Hou sewife
Others
21
74
280
105
80
14
20
56
185
78
I
96
129
55
.'
Frequency of" earillg OUI
Everyday
On ce a week
Two to three times a week
Two to three tim es CJ mont h
Once a month
Others
3.3
9.3
30.8
13
0.2
16
21.5
8.3
Cooking urensils
Stove
Mi c rowa ve
Oven
Grilled-stove
Grill ed charcoal
Othe rs
16.8
21.8
23.3
13 .2
10.2
14.7
104
17.3
13
22.2
17
22.7
7.8
133
102
136
47
592
265
22 1
64
98.7
44 .2
36.8
13.3
10.7
23
3.8
XO
Variabl e
of
food
frequency
Cronbach's Alpha
Food consumption frequ ency
Food consumption portio n size
Buy o r coo k th e fo od s
Pla ces of fo od avail a bility
0.730
0.634
0.902
0.840
3.5
12.3
46.7
17. 5
13.3
2.3
101
131
140
79
61
88
n
summary
8
HOl/se/wId income
::,,1000
1000- 1999
20002999
3000- 3999
4000--4999
:::5000
Tabl e 2. R eliability
que stionnaire.
( con lil1ued)
The percentage for ma le respondents was 50.5% ; for
female respondents it was 49.5%. There were 59.5%
Malay respondents, 29.8% Chinese, 10.2% Indian,
a nd 0.5°;;, of other ethnic (Table I). Overall, the
cook ing method fa pparatus of respondents was mostly
by stove (98.7 % ), followed by microwave (44.2%) and
oven (36.8 % ). The hi gher usage frequency of stove,
microwave and oven was probably due to their
con vellience. A gri lied stove ( 13.3 %) and grilled
charcoal (10.7%) were used less by respondents. On
the other hand , the mos tly used type of cooking was
stir-fried (65.7 % ) followed by deep-fried (41.3%),
boiling (34 % ) and baking (33.3%). This fact showed
that respondents preferred to consume fried foods
ＨｳｴｩｾＧＭｦｲ･､＠
and deep-fried) rather than other types of
food (Table I) . HCAs are toxic compounds formed
during grilling and frying of meat products in oil
(Jagerstad et al. 1998). The gri lli ng/barbecuing method
of food preparation has been shown to produce
significant level of HCAs (Keating and Bogen 2004).
In the United States, Sinha et al. (1995) reported the
highest level of PhlP in pan-fried or grilled/barbecued
chicken samples. In that study, grilled/ barbecued
chicken contained the highest levels of PhIP ranging
from 27 to 480 ngg - ' and pan-fried chicken contained
PhIP ranging from 12 to 70ngg- 1 In other studies in
the United States, Knize et al. (199 7) also reported
high level of PhIP (270 ng g-') in grilled chicken,
wherea s Keating and Bogen (2004) reported PhIP level
of 38.2 ng g-' in fried chicken and 327 .6 ngg- ' in
grilled chicken. In Spain, Busqucts et al. (2004)
reported the highes t level of PhIl' (46.9 ng g-') in
frjed chick en, whereas in Japa n, Wakabayashi et al.
(1993) PhlP was in fried codfish (69.2ngg- ') .
r
1064
M.H.A. Jahuru/ et al
(A)0.10-,--- -- - --
---------------------,
0.08
0.06
:::l
surc
to polycyclic aromatic hydrocarbons through consumption
of edible marin e spec ies in Catalonia, Spain. 1 Food Prot.
69:24932499.
!'vlarti-Cid R , Bacia A, Liobet 1M, Domingo JL. 2007. The
intake of chemical contaminants through fi sh and seafood
cons umpti on by children of Catalonia, Spain: heal th ri sks.
Food Chem T oxico!. 45: 1968- 1974.
Me ss ner C, Murk ovic M. 2004. Evaluation of a new model
sys tem for stud ying th e form a tion of heterocyclic amines.
J Chromatogr 13. 802: 19- 26.
Nowell S, Coles B, Sinha R, MacLeod S, Ratnasinghe DL.
Stotts C, Kadlubar FF. Arnbrosone CB . Lan g NP. 2002.
A nalysis of tota l meat intake a nd expos ure to individual
heteroc ycl ic amines in a case-co ntro l stud y of co lo rectal
ca ncer: co ntributi o n of m etabolic variation to ri sk.
Mutat Res. 506507 175185.
Olsson Y, Skog K , Lundstrom K. Jiigersta d M . 2005.
Color photographs for estimation of heterocyclic amine
intake from fried pork chops o f different RN genotypes
indi cate large varia tion s . Food Qual Prefer. 16:91 - 101.
Rohrrn a nn S, Becker N . 2002. D eve lopment of a short
questionnaire to assess the dietary intake of heterocyclic
aromatic amines . Publ Hlth Nutr. 5:699-75.
Sinha R , Chow WH , Kulldorff M . Dcnobilc .I , Butler J,
Garcia-Closas M. \Vei l R. Ho ove r RN, Rothman N. 1999.
Well-done gr ill ed red meat increases the ri sk o f colorectal
adenomas. Cancer Res . 59:43204324.
Sinha R. Gustafson DR, KulldorfT M , Wen WQ , Cerhen JR,
Zheng W. 2000. 2-Am in o- I-m eth yl-6-phe nyl imid azo[4 ,5bjpyridine, a carcinogen in hi gh tempera ture-coo ked meat
and breast can ce r ri sk. J Natl Cancer Imt. 92:1352- 1354.
Sinha R, Kulldorfr tv\. Cho\\ WH , Denobile J. Rothman N.
200 1. Di etary intake o f hcterocvc li c amines , meat-de ri ved
mutagenic activit y. and th e risk of colorcctal adenomas.
Cance r Ep id cmi ol Biomarkers Prevo 10:559- 562.
Sinha R, Kulldorff M , Curtin '/ , Brow n Cc, Alavanja MC,
Swanson CA 199X. Fri ed well -don e red meat and risk of
lun g cancer in wome n (United S tates). Cancer Causes
Control. 9:621630.
Sinha R, Kulldorfr M , Swan so n CA. C urtin J.
Brownso n RC. Alavanja MCR . 2000. Dietary heterocycl ic
amines and th e ri sk of lung cancer among Mi sso uri
wo men. Cancer Re s. 60:3753- 3756.
Sinh a R, Rothman N, Brown ED, Salmon CP, Kni ze MG,
Swanson CA. Ro ss i SC, Mark SO. Levander OA,
Felt on JS. 1995. Hi gh concen tra tion s o f th e carcin ogen
2-a mi no-I-mel hy 1-6-phcn ylim idazo-[4, 5-bi pyridi nc (Phi P)
occ ur in chicken but are dependent o n the cooking
meth od. Ca nce r Res. 55:4516--4519 .
Sinha R, Rothman N. Salmon CP, Knize MG, Brown ED,
Swanson CA Rhodes D , Rossi S. Felton 1S,
Levander OA. 1998. Heterocyc li c amine content in beef
cooked by different me thods to varying degrees of doneness and gravy made from meat drippings. Food Chem
Toxicol 36:279287.
Skog K. 199 3. Cooking procedure and food mutagens:
a literature review. Food Chem Toxicol. 31:655675.
Skog K , Augustsson K. Steineck G, Stenberg M ,
Jiigerstad M. 1997. Polar and non-polar heterocyclic
amines in cooked fi sh and mea t products and their
co rresponding pan residues . Food Chem Toxi co l.
35555-565.
Skog K , Solyakov A. 2002. H eterocycl ic amines in poultry
products: a literature rev iew. Food Chem Toxicol.
40 1213-1221.
Thomson BM. Lake RJ, Cressey PJ , Knize MG. 1996.
Est imat ed cancer risk from heterocycl ic amines in coo ked
meat :- a New Zealand perspective. Proc Nulr Soc NZ.
21: I 06115.
Wakaba yas hi K. Nagao M , Esumi 1-1 , Sugimura T. 1992.
Food-derived mutagens and carcinogens. Cancer Res.
52:S2092S2098.
Food Additives and Contaminan ts
Wakabayas hi K. T o tsuka Y, Fukuto me K . Oguri A,
Ush iyama H , Sugimura T. 1997. Human exposure to mutagenic/carcinogenic hete rocyclic a mines and co-mutagenic
fJ-carbolines. Muta t Res. 376:253259.
Wakabayashi K . U shi ya ma H , Takahashi M , Nukaya H ,
Kim S-B , Hiro se M . Ochiai M , Sugimura T , Nagao M .
1993. Exposure to hete rocyclic a mines. Environ Hlth
Perspect. 99:129- 133.
1071
Wong KY, Su J , Kni ze MG, Koh WP , Seow A . 2005.
Dieta[y exposure to heterocyclic am in es in a Chinese
pop ulation . NutI' Cancer. 52(2) :147- 155.
Z irnm erli B. Rhyn P, Zolle[ O . Schlatter J. 200 I . Occurrence
of he terocyclic aromatic amines in the Swiss diet: ana lytical method , exposure estimation and ri sk assess ment.
Food Addit Co ntam . 18:533- 55 1.
Vol. 27 , No.8 , August 201 0, 1060- 1071
Q
ｾ＠
Taylor &Francis
Taylor£..Fra nclsGroup
Dietary exposure to heterocyclic amines in high-temperature cpoked meat and fish in Malaysia
\.
."
'"
｣Ｌ ｾ＠
¥?
1
M.H.A. Jahurul, S. Jinap*, SJ. Ang, A. Abdul-Hamid, P . Hajeb. H.N. Lioe t and I.S.M. Zaidul
Cenl er of" Excellence /i)r Food S a/i' ly Research (C EFS R ) . Facultv
43400 U PM, S elango r, Malavsia
0/ Foo d Scien ce and Technology, Universili Putra Malaysia.
(R ecci l'cd ]5 janllan ' 20 11!. /il1a l I'crsiun received 2] M arch 20 10 )
The inta ke of hcterocycl ic amin es is influenced by th e am ount and type of mca t and fish inges ted, frequen cy of
consumpti o n, cooking method s. cooking tem pe rature, and du rati on of cooking. In thi s study, the dietary intake
o f heterocyclic amincs in Ma lays ia and th eir main sources were inves ti gated . Fo rt y-two samples of meat and fi sh
were anal ysed by hi gh-perfo rm a nce liquid chromatograp hy with pho todi ode array detector to determin e
the concent rati on of th e six predomin a nt heterocyclic amin cs, namely: 2-amino-3-meth ylimidazo[4,5f]q uin ol ine (I Q ). 2-a mi no-3 A-dimet hylimidazo[4. 5-f lq uino Ii nee 1\1 eIQ). 2-amino- 3,S-dimethylimidazo[4 ,5･ｴ ｨ ｹ ｬｩｭ､
｡ｺｯ
｛ ＴＬＵＭ
ｦ＠ Jquinoxalin e (4,8- DiMeIQx), 2-amino-3 ,7,Sflq uin ox alin c (M e l Qx) . 2-amin o- Ｓ ａ ＬｾＭ ｴｲｩｭ
trim eth yl imida w [4. 5-1' Jq uin oxalin e (7 .S-Di Me l Qx). ,1 nd 2·am ino - I -mcth yl-6-phcnylimidazo[4,5-b]pyridinc
(PhIP). Dietary intak e dat a we re obtained lI sing a food-freq uen cy qu es ti o nn a ire wh en interviewing 600
Ma la ys ia n res pondents. The level of tota l heterocyclIc ami nes in food sam ples studies ranged fr om not detected
to 38.7 ng g- I. T he a ve ra ge dail y intake level of hete ro cycl ic am ine was 553.7 ng per capita day- I. The intake of
PhIP was th e hig hest. followed by MeI Qx and Ivl eIQ. T he result s revea l tha t fri ed a nd grill ed chicken were the
maj or di eta ry so urce o f hetewcycl ic am ines ill ivlalaysia . However. the heterocycl ic a mine intake by the'
Mala ys ian popu lati on was lo \\'c r tha n th ose repor ted fr olll other reg ions
Keywords: exposllt'c a,;sesslll cnt: heterocycli c am in es: co oked fo od,;
Introduction
Heterocycli c a mines (HCAs) a re possible human
a nd animal carcin oge n s fo nned in cooked m eat an d
fi sh during hi g h-temp e ra ture c ookin g processes
(Internation a l A ge ncy for R esea rc h o n Cance r
(IARC) 1993). Th e rAR C a lso reported that
HCAs such a s Ｒ Ｍ ｾ［ ｭｩｮｯ
ＭＳＬ
ＴＭ､ｩｬＱ･ｴｨ
ｹ ｬｩ､｡
ｺｯ ｬＴ Ｎ ＵＭ
fJquinoline,. (MelQ), 2-amino-3,8-dime th ylimd azo [4,5fJquinoxalin e (MeIQx), and 2-a mino-l-meth yl-6-phen ylimidazo [4, 5-b) pyridine (PhIP) are possible huma n
carcinogens;
and
2-am ino-3-me th ylimida zo[4,5-f]quinoline (IQ) a s a probabl e ca rcinogen. H C A s a lso
ca use cancer at a varie ty of multiple sites in labo ratory
a nimal species (EI-Ba y oum y et al. 1995), Several ca secontrol studies in various p o pula tion s h ave repo rted
positive associati o n s between a highe r intake of wellcoo ked meat and th e risk of colon cancer (Sinh a e t al.
1999), breast ca ncer (Sinha e t a l. 2000), lun g ca nce r
(Sinha 1998), and gas tric cance r (de Stefani et a l. 1998).
Huma n s arc ex p o sed to p o te nti a ll y to x ic s u bstances
including natura lly occ urrin g muta geni c and carci n o ge nic c o mpoun ds . On e's daily di e t lll ay c o nt ai n H C A s
a nd polycyclic a romatic hydroca rbon s ( PAH s) as a
res ult of high-temperature processi ng o f m eat a nd fi sh .
Bo t h gro ups a re eas il y found in grilled and fri ed meat,
p o ultry an d fi sh (Gomaa et al. 199 3; Kaze rouni e t al.
200 I; J iigersta d a nd Skog 20(5), T he present study is
foc llsed on H C A s. Friedman (1996) reported that some
H CA s pl ay a n im p o rtant rol e in th e taste, fla vour, a nd
eve n smell o f h ea ted meat. H C A s are form ed by th e
pyro lys is of pro te in a nd amino acids in pro tein-rich
food s. Th e ir form a tion depend s on several factor s such
a s tempe rature, c ooking method and time, the degree
of d o ne ness, th e c oncentration of HCA prec ursors, and
th e presence of co mpounds with enhancing or inhibit o ry e ffects (Felto n et al. 2000; Busquets et a L 2004) .
There has bee n a large variability of estimated intake
of di eta ry H C A s reported from different countries
(suc h as Japan , Singapore , Sweden , Switzerland ,
Spa in , th e Unite d Kingdom, a nd the United States).
The rea so n ma y be due to diffe rent cooking method s,
cook in g te mperature. duration of cooking, different
ques tio nnaire m a ke-up, and th e studied populations .
ｈ ｯ｜Ｇ
･ ｜｣ｾ ｲＬ＠ fo r th e in ves tigation o f hum a n ca rcinogen s,
an a cc ur a te assess m e nt of th e indi vidua l co nsumption
of H eAs is esse nt ia l. U p-to-dat e . exte nsive research on
HCA s has been p e rfo rm ed on th e W es tern die t
(Wakabay a shi et a l. 1992; S kog 199 3; Kni ze et al.
1995: Z il11ll1c rli et a l. 2001: Sk og a nd Sol ya kov 2002 ;
Ｊｃ ｯ ｲ ･ｳｾ
ｮ､ｩ ｧ＠
author. Emails:jinap@ food.upm.edu .my:sjinap@gmail.co m
' Present address : Depa rtm ent of Food Science an d Tec hn ology, Bogor Agri cultura l University, Bogo r, Indonesia
ISSN 0265-203X pri nt/lSS N 1464-51 22 o nl ine
ro 10 10 Tayl or & Francis
DO l: tO I080i]944004 1003801 190
http: // ww w.in fo rillaw orld .com
Fo od Additives and Contaminants
Cantwell et ai. 2004; Llobet et a!. 2006: Domingo et a!.
2007; Marti-Cid et ai. 2007). Fried meat is an important source of exposure to HCAs in the Western diet.
Although HCAs are formed at ppb levels (ngg- I),
their intake may be dangerous for human health
because of the total amount of cooked meat or fish
consumed (Busquets et a1. 2004).
This is the first extensive study on HCA exposure in
Malaysia. The analysis of Malaysian foods for the
occurrence of HCAs is important because there is
worldwide human exposure to these compounds
according to many reports (Augustsson et a!. 1997:
Byrne et ai. 1998; Kobayashi et ai. 2002; Wong et ai.
2005). The aim of the present study was to determine
the level of major six HCAs - IQ, MeIQ, MelQx, 4, 8DiMelQx, 7, 8-DiMeIQx and PhIP - in some of the
most commonly consumed foods in Malaysia in order
to estimate the exposure of the Malaysian population
to these toxic compounds. The assessment was performed by combining the content of HCAs in cooked
food samples analysed in the laboratory with information on food consumption from the food frequency
questionnaire.
Materials and methods
Study population and questionnaire
A cross-sectional survey was conducted in the central
zone of Peninsular Malaysia covering three locations:
Kuala Lumpur, Putra Jaya, and Selangor state. The
respondents comprised three main races: Malay
(59.5%), Chinese (29.8%), . and Indian (10.2%) , and
other ethl/ie (0.5%), which were almost similar in
proportion to the Malaysian population. A total
sample size of 600 adult respondents aged IS years
and above was selected randomly for the survey. One
adult was chosen to represent the pattern of the whole
household. The sample size was measured by using a
two-staged stratified random sampling method and the
following equation was applied in the sample size
estimation:
ｎ
-Ｍｾ
1.96 2 x P(I - P)
de
(I)
where a Z-value of 1.96 for p=0.05 (95% confidence
level); P is the estimated prevalence of the sample equal
to 0.5; and d is a margin of error of 0.1 for ± I 0%.
In the first stage of stratified random sampling, 75
enumeration blocks were selected from living quarters
where each living quarter consists of between 80 and
120 houses. In the second stage of stratified random
sampling, eight households were selected from each
enumeration block. The enumeration blocks and living
quarters for the survey were provided by the
Department of Stati stics Malaysia .
1061
Study design and questiollnaire
The questionnaire was divided into three sections:
cooking utensils and cooking methods used at home;
demographic data of the respondent; and dietary
intake. Dietary intake was assessed using a food
frequency questionnaire with a 1-month recall. The
food frequency questionnaire included information on
food items, the frequency of food consumption and the
portion size of food ingested. Before the main survey,
a pilot survey with 60 respondents was performed in
the targeted areas to test question planning in the
questionnaire.
Selection o/food items and samplillg offoods
Food item s were selected based on the literature
publi shed in peer-revievied journals, which included
all types of piocessed (such as drying and smoking)
and cooked (such as grilling, roasting and frying) foods
report ed to contain !-leAs . There were in total 42 types
of food item s; three samples were purchased from the
same stall at different times of the day. The selected
meat and fish dishes for this study were fried fish, fried
chicken (deep-fried), chicken soup , chicken curry,
steamed fi sh. chi cke n cooked with chilli (masak
merah). chicken burger, {ish curry, fried chicken,
keropok kkor, chicken satay. chicken randang, fish
fried in chilli, nu gge t, fish in tamarind (asam pedas
Ikan), paprika chicken, sweet-and-sour fish, sausage,
fish soup , chicken in coconut milk gravy (ayam masak
lemak), fri ed salted fish, fish in coconut milk, chicken
percik (ayalll percik), fried beef, beef cooked with
chilli, roasted fish, beef curry, smoke fish, fish in soya
sa uce , beef burger, beef satay, beef rendang, chicken
golek (ayam golek), fish in tauClI sauce, beef soup,
chicken kurma, mutton satay, paprika beef, fish with
three navours , grilled chicken tandoori, mutton curry,
beef in coconut milk gravy (masak lemak daging),
barbecue-grilled chicken, black pepper-grilled chicken,
beef kurilla , and honey-grilled chicken.
Samples of all food items were purchased from
local food stalls in Kuala Lumpur, Selangor, and
Putrajaya. Upon arrival in the laboratory, samples'
bones were removed from the meat and fish , then
samples were homogenized using a Waring blender
(Dawsonville, GA , USA). The homogenized samples
were then pooled and mixed together before being
quarter sampled. The representative samples were
stored at -18 v C until analysis.
Statistical analysis
Statistical analyses were performed using SPSS
(version 14.0). The reliability analysis was measures
as an internal consistency. Cronbach's alpha was the
measurement for internal consistency. The survey was
1062
Af.H.A. Jahllrlll et aJ.
considered to be reliable if Cronbach's alpha was at
least 0.7. The descriptive analysis, including the mean,
median, mode, standard deviation, and frequency of
food taken by respondents, was used to describe the
basic features of the data collected from the survey. An
independent sample {-test was used to compare the
differences between two genders. Analysis of variance
(ANOVA) was used to test the differences between
different HCAs in different food items. A p < 0.05 was
considered to be statistically significant.
Chemicals
Solvent and chemicals were of both high-performance
liquid chromatography (HPLC) and analytical grade.
The water was purified in an Elix-MilliQ system
(Millipore, Bedford, MA, USA). All solutions were
passed through a 0.45-l1m filter before injection into
the HPLC system. HCAs standards, i.e . 2-amino-3methylimidazo[4,5-f]quinoline
(lQ),
2-amino-3,4dimethylimidazo[4,5-f]quinoline (MelQ), 2-amino3 ,8-dimeth y Iimidazo[ 4, 5-f]q uinoxaline
(Mel Qx),
2-ami no- 3,4,8- trimethylimidazo[ 4,5-f]quinoxaline (4,8DiMeIQx),
2-amino-I-methyl-6-phenylimidazo[4,5b]pyridine
(Ph I P),
2-amino- 3, 7 ,8-trimethylimidazo[4,5-f]qunoxaline (7,8-DiMeIQx), and 2-a111ino3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline
(4,7,8TriMelQx) as internal standard, were purchased from
Toronto Research Chemicals (Dowl1sview. ON,
Canada). Empty Extrelut-20 extraction cartridges
were provided by Merck (Darmstadt, Germany) and
Oasis MCX cartridges (3 mli60 mg) by Waters
(Milford , MA, USA). MCX cartridges were preconditioned with ethyl acetate (2 ml). The solute
diatomaceo,us earth refill material was obtained from
1ST (Hengoed, UK).
RCA extraction
The extraction and clean-up of all six HCAs studied
was carried out following the modified method of
Gross and Griiter (1992) and Messner and Murkovic
(2004). Samples (I g) spiked with internal standard
(4,7,8-TriMcIQx, 250 ppb) were dissolved in 12ml of
1 M NaOH and the suspension was homogenized using
magnetic stirring for 3 h with 500 rpm (Heidolph, MR
3001 K). The alkaline solution was mixed with diatomaceolls earth (13 g) , and then transferred into empty
Extrelut columas. Ethyl acetate was used as the
extraction solvent and elute was passed through the
coupled Oasis MCX cartridges. The MCX cartridges
were washed with 0.1 M HCI (2 ml) and MeOH
(2 ml). The analytes were eluted with 2 ml of
MeOH-concentrated ammonia (19/1, vjv). The samples were evaporated to dryness under a stream of
nitrogen and the final extracts were dissolved in 100 pi
methanol just before analysis for HCAs. Recovery of
HCAs was determined using the standards addition
method. The standards mixture (200 and 500 ng g- I
sample) was spiked to the chicken, beef, and fish
samples before the extraction step. HCAs were separated using reversed-phase LC and a TSK gel ODS T M
column (25 em x 4.6 mm. 5 11m). The LC systems consisted of a Waters LC model 2690 (CA, USA) with a
photo-diode array detector (Waters model 2996),
equipped with a quaternary solvent delivery system
under a Waters Controller model 600, an autosampl er
(Waters model 717) , and an on-line vacuum degasser.
The mobile phase consisted of solvent A, 0.0 I M
triethylamine in water (adjusted to pH 3.3 with acetic
acid), and solvent B, acetonitrile (HPLC grade;
Merck). Analyses were carried out in a !!radient
mode for 33 min. Gradient programming \;as performed as follows: at zero to 18 min it was 5% to 23%
B in A, at 18- 25 min it was 23% B in A, and at
25--33 min it was 23% to 25% B in A. The flow rate of
the mobile phase was I ml min - I.
Determination of LOD and LOQ
The limit of detection (LOD) and limit of quantification (LOQ) were obtained by performing seven determinations of the lowest acceptable standard
concentration. LOD was calculated as three times the
standard of deviation (SD); LOQ was ten times the SD
of the average background signal.
Estimated dai{.v intake
The estimated daily intake levels of HCAs by an adult
(ng day - I per capita) were calculated using the average
of each meat and fish product, i.e, (Hajeb et a!. 2009):
Daily intake per person per day]
= mean concentration (ngg- I) of HCAs x amount of
RccIJl'er), test
RecO\ery assays were established by spiking known
amounts of the matrices (200 and 500 ng g - I sample) of
the HCA standard solution to samples. Seven replicates
were conducted on each fortified sample. The recovery
assays were calculated by comparing the concentration
of the fortified samples with those of the unfortified
samples.
meat and fish products consumed per day (g per capita)
Results 'and discussion
The socio-demographic characteristics of the study
population are summarized in Table I. The distribution for the gender and the ethnic group of the
respondents represented the Malaysian population.
Food Additives and Contaminants
Table I. Socio-demographic chara cteri stics of respondents.
Tabl e 1. Continued .
Demographic profil e
Demographic profil e
Frequency
%
Gender
Male
Female
303
297
50.5
49.5
Elhniciry
Malay
Chinese
Indian
Othe rs
357
179
61
3
62.5
29.8
10.2
0.5
18- 24
25-29
30-34
35- 39
40--44
45--49
50- 54
55- 59
6064
130
91
64
64
67
52
48
30
22.2
15.2
10.7
10.7
11.2
8.7
65- 69
20
5
3.0"
3.3
ｾＷＰ＠
II
1.8
1063
Frequency
%
394
248
204
200
249
255
65.7
41.3
34
33.3
41.5
42.5
Cooking merhod
Stir-fried
Deep-fried
Boiling
Baking
Grilled
Roasted
Age group (rears )
23
Educa rion /('1'1'/
Uneducated
Primar y sc hoo l
Seco nd ary scho ol
College
Bachelor deg ree
Postgrad uCJt e
Others
Carccr categories
Government se r va nt
Pri va te sec to r
Self-employed
Farmer
Student
Hou sewife
Others
21
74
280
105
80
14
20
56
185
78
I
96
129
55
.'
Frequency of" earillg OUI
Everyday
On ce a week
Two to three times a week
Two to three tim es CJ mont h
Once a month
Others
3.3
9.3
30.8
13
0.2
16
21.5
8.3
Cooking urensils
Stove
Mi c rowa ve
Oven
Grilled-stove
Grill ed charcoal
Othe rs
16.8
21.8
23.3
13 .2
10.2
14.7
104
17.3
13
22.2
17
22.7
7.8
133
102
136
47
592
265
22 1
64
98.7
44 .2
36.8
13.3
10.7
23
3.8
XO
Variabl e
of
food
frequency
Cronbach's Alpha
Food consumption frequ ency
Food consumption portio n size
Buy o r coo k th e fo od s
Pla ces of fo od avail a bility
0.730
0.634
0.902
0.840
3.5
12.3
46.7
17. 5
13.3
2.3
101
131
140
79
61
88
n
summary
8
HOl/se/wId income
::,,1000
1000- 1999
20002999
3000- 3999
4000--4999
:::5000
Tabl e 2. R eliability
que stionnaire.
( con lil1ued)
The percentage for ma le respondents was 50.5% ; for
female respondents it was 49.5%. There were 59.5%
Malay respondents, 29.8% Chinese, 10.2% Indian,
a nd 0.5°;;, of other ethnic (Table I). Overall, the
cook ing method fa pparatus of respondents was mostly
by stove (98.7 % ), followed by microwave (44.2%) and
oven (36.8 % ). The hi gher usage frequency of stove,
microwave and oven was probably due to their
con vellience. A gri lied stove ( 13.3 %) and grilled
charcoal (10.7%) were used less by respondents. On
the other hand , the mos tly used type of cooking was
stir-fried (65.7 % ) followed by deep-fried (41.3%),
boiling (34 % ) and baking (33.3%). This fact showed
that respondents preferred to consume fried foods
ＨｳｴｩｾＧＭｦｲ･､＠
and deep-fried) rather than other types of
food (Table I) . HCAs are toxic compounds formed
during grilling and frying of meat products in oil
(Jagerstad et al. 1998). The gri lli ng/barbecuing method
of food preparation has been shown to produce
significant level of HCAs (Keating and Bogen 2004).
In the United States, Sinha et al. (1995) reported the
highest level of PhlP in pan-fried or grilled/barbecued
chicken samples. In that study, grilled/ barbecued
chicken contained the highest levels of PhIP ranging
from 27 to 480 ngg - ' and pan-fried chicken contained
PhIP ranging from 12 to 70ngg- 1 In other studies in
the United States, Knize et al. (199 7) also reported
high level of PhIP (270 ng g-') in grilled chicken,
wherea s Keating and Bogen (2004) reported PhIP level
of 38.2 ng g-' in fried chicken and 327 .6 ngg- ' in
grilled chicken. In Spain, Busqucts et al. (2004)
reported the highes t level of PhIl' (46.9 ng g-') in
frjed chick en, whereas in Japa n, Wakabayashi et al.
(1993) PhlP was in fried codfish (69.2ngg- ') .
r
1064
M.H.A. Jahuru/ et al
(A)0.10-,--- -- - --
---------------------,
0.08
0.06
:::l
surc
to polycyclic aromatic hydrocarbons through consumption
of edible marin e spec ies in Catalonia, Spain. 1 Food Prot.
69:24932499.
!'vlarti-Cid R , Bacia A, Liobet 1M, Domingo JL. 2007. The
intake of chemical contaminants through fi sh and seafood
cons umpti on by children of Catalonia, Spain: heal th ri sks.
Food Chem T oxico!. 45: 1968- 1974.
Me ss ner C, Murk ovic M. 2004. Evaluation of a new model
sys tem for stud ying th e form a tion of heterocyclic amines.
J Chromatogr 13. 802: 19- 26.
Nowell S, Coles B, Sinha R, MacLeod S, Ratnasinghe DL.
Stotts C, Kadlubar FF. Arnbrosone CB . Lan g NP. 2002.
A nalysis of tota l meat intake a nd expos ure to individual
heteroc ycl ic amines in a case-co ntro l stud y of co lo rectal
ca ncer: co ntributi o n of m etabolic variation to ri sk.
Mutat Res. 506507 175185.
Olsson Y, Skog K , Lundstrom K. Jiigersta d M . 2005.
Color photographs for estimation of heterocyclic amine
intake from fried pork chops o f different RN genotypes
indi cate large varia tion s . Food Qual Prefer. 16:91 - 101.
Rohrrn a nn S, Becker N . 2002. D eve lopment of a short
questionnaire to assess the dietary intake of heterocyclic
aromatic amines . Publ Hlth Nutr. 5:699-75.
Sinha R , Chow WH , Kulldorff M . Dcnobilc .I , Butler J,
Garcia-Closas M. \Vei l R. Ho ove r RN, Rothman N. 1999.
Well-done gr ill ed red meat increases the ri sk o f colorectal
adenomas. Cancer Res . 59:43204324.
Sinha R. Gustafson DR, KulldorfT M , Wen WQ , Cerhen JR,
Zheng W. 2000. 2-Am in o- I-m eth yl-6-phe nyl imid azo[4 ,5bjpyridine, a carcinogen in hi gh tempera ture-coo ked meat
and breast can ce r ri sk. J Natl Cancer Imt. 92:1352- 1354.
Sinha R, Kulldorfr tv\. Cho\\ WH , Denobile J. Rothman N.
200 1. Di etary intake o f hcterocvc li c amines , meat-de ri ved
mutagenic activit y. and th e risk of colorcctal adenomas.
Cance r Ep id cmi ol Biomarkers Prevo 10:559- 562.
Sinha R, Kulldorff M , Curtin '/ , Brow n Cc, Alavanja MC,
Swanson CA 199X. Fri ed well -don e red meat and risk of
lun g cancer in wome n (United S tates). Cancer Causes
Control. 9:621630.
Sinha R, Kulldorfr M , Swan so n CA. C urtin J.
Brownso n RC. Alavanja MCR . 2000. Dietary heterocycl ic
amines and th e ri sk of lung cancer among Mi sso uri
wo men. Cancer Re s. 60:3753- 3756.
Sinh a R, Rothman N, Brown ED, Salmon CP, Kni ze MG,
Swanson CA. Ro ss i SC, Mark SO. Levander OA,
Felt on JS. 1995. Hi gh concen tra tion s o f th e carcin ogen
2-a mi no-I-mel hy 1-6-phcn ylim idazo-[4, 5-bi pyridi nc (Phi P)
occ ur in chicken but are dependent o n the cooking
meth od. Ca nce r Res. 55:4516--4519 .
Sinha R, Rothman N. Salmon CP, Knize MG, Brown ED,
Swanson CA Rhodes D , Rossi S. Felton 1S,
Levander OA. 1998. Heterocyc li c amine content in beef
cooked by different me thods to varying degrees of doneness and gravy made from meat drippings. Food Chem
Toxicol 36:279287.
Skog K. 199 3. Cooking procedure and food mutagens:
a literature review. Food Chem Toxicol. 31:655675.
Skog K , Augustsson K. Steineck G, Stenberg M ,
Jiigerstad M. 1997. Polar and non-polar heterocyclic
amines in cooked fi sh and mea t products and their
co rresponding pan residues . Food Chem Toxi co l.
35555-565.
Skog K , Solyakov A. 2002. H eterocycl ic amines in poultry
products: a literature rev iew. Food Chem Toxicol.
40 1213-1221.
Thomson BM. Lake RJ, Cressey PJ , Knize MG. 1996.
Est imat ed cancer risk from heterocycl ic amines in coo ked
meat :- a New Zealand perspective. Proc Nulr Soc NZ.
21: I 06115.
Wakaba yas hi K. Nagao M , Esumi 1-1 , Sugimura T. 1992.
Food-derived mutagens and carcinogens. Cancer Res.
52:S2092S2098.
Food Additives and Contaminan ts
Wakabayas hi K. T o tsuka Y, Fukuto me K . Oguri A,
Ush iyama H , Sugimura T. 1997. Human exposure to mutagenic/carcinogenic hete rocyclic a mines and co-mutagenic
fJ-carbolines. Muta t Res. 376:253259.
Wakabayashi K . U shi ya ma H , Takahashi M , Nukaya H ,
Kim S-B , Hiro se M . Ochiai M , Sugimura T , Nagao M .
1993. Exposure to hete rocyclic a mines. Environ Hlth
Perspect. 99:129- 133.
1071
Wong KY, Su J , Kni ze MG, Koh WP , Seow A . 2005.
Dieta[y exposure to heterocyclic am in es in a Chinese
pop ulation . NutI' Cancer. 52(2) :147- 155.
Z irnm erli B. Rhyn P, Zolle[ O . Schlatter J. 200 I . Occurrence
of he terocyclic aromatic amines in the Swiss diet: ana lytical method , exposure estimation and ri sk assess ment.
Food Addit Co ntam . 18:533- 55 1.