heterocyclicaromaticaminesincookedmeat products:causes...

Heterocyclic Aromatic Amines in Cooked MeatProducts: Causes, Formation, Occurrence, andRisk AssessmentMonika Gibis

Abstract: Meat products are sources of protein with high biological value and an essential source of other nutrients,such as vitamins and minerals. Heating processes cause food to become more appetizing with changes in texture,appearance, flavor, and chemical properties by the altering of protein structure and other ingredients. During heattreatment, heterocyclic aromatic amines (HAAs), potent mutagens/carcinogens, are formed due to the Maillard reaction.The HAAs are classified in at least 2 groups: thermic HAAs (100 to 300 °C) and pyrolytic HAAs (>300 °C). This reviewfocuses on the parameters and precursors which affect the formation of HAAs: preparation, such as the marinating ofmeat, and cooking methods, including temperature, duration, and heat transfer, as well as levels of precursors. Additionally,factors are described subject to pH, and the type of meat and ingredients, such as added antioxidants, types of carbohydratesand amino acids, ions, fat, and other substances inhibiting or enhancing the formation of HAAs. An overview of thedifferent analytical methods available is shown to determine the HAAs, including their preparation to clean up thesample prior to extraction. Epidemiological results and human daily intake of HAAs obtained from questionnaires showa relationship between the preference for very well-done meat products with increased HAA levels and an enhancedrisk of the incidence of cancer, besides other carcinogens in the diet. The metabolic pathway of HAAs is governed bythe activity of several enzymes leading to the formation of DNA adducts or HAA excretion and genetic sensitivity ofindividuals to the impact of HAAs on human cancer risk.

Keywords: antioxidants, β-carbolines, heterocyclic aromatic amines, meat, precursors

IntroductionIndividuals have been exposed to a range of toxic substances

throughout human history. These can be naturally occurring mu-tagens, which are mainly found in plant substances, or process-induced mutagens, which can arise during manufacture, such asfrom heating. Nitrosamines, polycyclic aromatic hydrocarbons,and heterocyclic aromatic amines are typical heat-induced com-pounds (Ferguson 2010; Oostindjer and others 2014). Widmark(1939) reported for the first time that extracts of roasted horse meatinduced cancer in the mammary glands of mice when multiple-swabbed on the back. The heterocyclic aromatic amines (HAAs)that are the focus of this article belong to the process-inducedmutagens that cannot be detected in unheated products (Skog andothers 1998b). In general, HAAs are formed from heated prod-ucts which contain sources of nitrogenous compounds, mainlyheated foods of animal origin, such as proteins and creatine (Skogand others 1998b). The formation of HAAs is principally de-pendent on the temperature, heat transfer, and heating conditions

MS 20151495 Submitted 3/9/2015, Accepted 1/12/2015. Author Gibis is withDept. of Food Physics and Meat Science, Inst. of Food Science and Biotechnology, Univ.of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany. Direct inquiries toauthor Gibis (E-mail: [email protected]).

used (Murkovic 2004a; Sugimura and others 2004). The struc-tures of HAAs which are formed at temperatures between 100 and300 °C are called thermic HAAs, IQ-type HAAs (imidazoquino-line or imidazoquinoxaline) or aminoimidazoazaarenes (Jägerstadand others 1998). Above 300 °C, pyrolysis of proteins and individ-ual amino acids occurs and HAAs are formed which are knownas pyrolytic HAAs or non-IQ-type HAAs (Jägerstad and others1998). Another classification of HAAs subdivides them into non-polar and polar HAAs (IQ-type compounds) due to their chem-ical properties. The names, abbreviations, properties, mutagenic-ity (Ames test), and molecular structures of HAAs are shown inTable 1.

Occurrence of Heterocyclic AminesBesides the important formation factors of cooking temperature

and duration of the heat treatment, thermic HAAs occur in almostall heated foods of animal origin, such as meat and fish, becausecreatine, the precursor needed for their formation, is containedin these products (Jägerstad and others 1998; Murkovic 2000).2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) hasbeen detected not only in products of animal origin, but alsoin wine, beer (Manabe and others 1993), and smoked cheese(Skog and others 1994; Naccari and others 2009). The sourceis possibly environmental, since PhIP can occur in burning

C© 2016 Institute of Food Technologists®doi: 10.1111/1541-4337.12186 Vol. 15, 2016 � Comprehensive Reviews in Food Science and Food Safety 269

Heterocyclic amines in cooked meat products . . .

Tabl

e1–

Chem

ical

stru

ctur

e,na

me,

and

mol

ecul

arw

eigh

tof

30H

AA

s;so

me

ofth

eir

prop

erti

esan

din

dica

tion

ofth

eir

mut

agen

icit

y(A

mes

assa

yin

the

pres

ence

ofS-

9m

ixus

ing

Salm

onel

laTy

phim

uriu

mst

rain

sTA

98,T

A10

0an

dTA

1538

.

Stru

ctur

eA

bbre

viat

ion/

Chem

ical

nam

eCA

SN

oM

olec

ular

wei

ght

(g/

mol

)Pr

oper

ties

aM

utag

enic

ity

(×10³r

ev/μ

g)b

2-A

min

o-im

idaz

oqui

nolin

esIQ 2-

Am

ino-

3-m

ethy

limid

azo[

4,5-

f]qu

inol

ine

7618

0-96

-6

192.

2po

lar

pKa

5.86

±0.

40c

433

(TA

98)7

(TA

100)

300

(TA

1538

)

Iso-

IQ2-

Am

ino-

1-m

ethy

limid

azo[

4,5-

f]qu

inol

ine

1024

08-2

5-3

192.

2po

lar

pKa

5.99

±0.

40c

na

MeI

Q2-

Am

ino-

3,4-

dim

ethy

limid

azo[

4,5-ƒ]

quin

olin

e77

094-

11-2

212.

3po

lar

pKa

6.22

±0.

40c

661

(TA

98)3

0(T

A10

0)85

9(T

A15

38)

2-A

min

o-im

idaz

oqui

noxa

lines

IQx

2-A

min

o-3-

met

hylim

idaz

o[4,

5-ƒ]

quin

oxal

ine

1083

54-4

7-8

199.

3po

lar

pKa

1.96

±0.

50c

75.4

(TA

98)1

.5(T

A10

0)10

3(T

A15

38)

8-M

eIQ

x2-

Am

ino-

3,8-

dim

ethy

limid

azo[

4,5-ƒ]

quin

oxal

ine

7750

0-04

-0

213.

3po

lar

pKa

2.20

±0.

50c

145

(TA

98)1

4(T

A10

0)84

.7(T

A15

38)

(Con

tinu

ed)

270 Comprehensive Reviews in Food Science and Food Safety � Vol. 15, 2016 C© 2016 Institute of Food Technologists®

Heterocyclic amines in cooked meat products . . .Ta

ble

1–Co

ntin

ued.

Stru

ctur

eA

bbre

viat

ion/

Chem

ical

nam

eCA

SN

oM

olec

ular

wei

ght

(g/

mol

)Pr

oper

ties

aM

utag

enic

ity

(×10³r

ev/μ

g)b

4-M

eIQ

x2-

Am

ino-

3,4-

dim

ethy

limid

azo[

4,5-ƒ]

quin

oxal

ine

1083

54-4

8-9

213.

3po

lar

pKa

2.32

±0.

50c

1162

(TA

98)5

1(T

A10

0)10

42(T

A15

38)

4,8-

DiM

eIQ

x2-

Am

ino-

3,4,

8-tr

imet

hylim

idaz

o[4,

5-ƒ]

quin

oxal

ine

9589

6-78

-9

227.

3po

lar

pKa

2.56

±0.

50c

183

(TA

98)8

(TA

100)

225

(TA

1538

)

7,8-

DiM

eIQ

x2-

Am

ino-

3,7,

8-tr

imet

hylim

idaz

o[4,

5-ƒ]

quin

oxal

ine

9218

0-79

-5

227.

3po

lar

pKa

2.49

±0.

50c

163

(TA

98)9

.9(T

A10

0)18

9(T

A15

38)

TriM

eIQ

x2-

Am

ino-

3,4,

7,8-

tetr

aim

ethy

limid

azo[

4,5-ƒ]

quin

oxal

ine

1328

98-0

7-8

241.

3po

lar

pKa

2.85

±0.

50c

na

4-CH

2O

H-8

-MeI

Qx

2-A

min

o-4-

hydr

oxym

ethy

l-3,8

-dim

ethy

limid

azo[

4,5-ƒ]

quin

oxal

ine

1539

54-2

9-1

243.

3po

lar

pKa

2.17

±0.

50c

13.5

2±

0.50

d

99(T

A98

)2.6

(TA

100)

(Con

tinu

ed)

C© 2016 Institute of Food Technologists® Vol. 15, 2016 � Comprehensive Reviews in Food Science and Food Safety 271


Tabl

e1–

Cont

inue

d.

Stru

ctur

eA

bbre

viat

ion/

Chem

ical

nam

eCA

SN

oM

olec

ular

wei

ght

(g/

mol

)Pr

oper

ties

aM

utag

enic

ity

(×10³r

ev/μ

g)b

7-M

eIgQ

x2-

Am

ino-

1,7-

dim

ethy

l-1H

-imid

azo[

4,5-

g]qu

inox

alin

e93

4333

-16-

1

213.

2po

lar

pKa

3.95

±0.

50c

na

7,9-

MeI

gQx

2-A

min

o1,7

,9-t

ridim

ethy

l-1H

-imid

azo[

4,5-

g]qu

inox

alin

e15

6243

-39-

9

227.

3po

lar

pKa

4.65

±0.

50c

0.67

(TA

98)

2-A

min

o-im

idaz

opyr

idin

esPh

IP2-

Am

ino-

1-m

ethy

l-6-p

heny

l-im

idaz

o[4,

5-b]

pyrid

ine

1056

50-2

3-5

224.

3po

lar

pKa

7.72

±0.

30c

1.8

(TA

98)0

.12

(TA

100)

2.9

(TA

1538

)

4’O

H-P

hIP

2-A

min

o-1-

met

hyl-6

-(4’h

ydro

xyph

enyl

)-im

idaz

o[4,

5-b]

pyrid

ine

1268

61-7

2-1

240.

6po

lar

pKa

7.79

±0.

30c

8.27

±0.

15d

0.00

2(T

A98

)

DM

IP2-

Am

ino-

1,6-

dim

ethy

limid

azo[

4,5-

b]py

ridin

e13

2898

-04-

5

162.

2po

lar

pKa

8.16

±0.

30c

0.00

8(T

A15

38)

(Con

tinu

ed)



Tabl

e1–

Cont

inue

d.

Stru

ctur

eA

bbre

viat

ion/

Chem

ical

nam

eCA

SN

oM

olec

ular

wei

ght

(g/

mol

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oper

ties

aM

utag

enic

ity

(×10³r

ev/μ

g)b

1,5,

6TM

IP2-

Am

ino-

1,5,

6-tr

imet

hylim

idaz

o[4,

5-b]

pyrid

ine

1610

91-5

5-0

176.

2po

lar

pKa

8.66

±0.

30c

100

(TA

1538

)

3,5,

6TM

IP2-

Am

ino-

3,5,

6-tr

imet

hylim

idaz

o[4,

5-b]

pyrid

ine

5766

7-51

-3

176.

2po

lar

pKa

8.36

±0.

30c

na

IFP

2-A

min

o-1,

6-di

met

hyl-f

uro[

3,2-

e]im

idaz

o[4,

5-b]

pyrid

ine

3573

83-2

7-8

202.

3po

lar

pKa

7.52

±0.

40c

̴10(T

A15

38)

α-C

arbo

lines

Aα

C2-

Am

ino-

9H-p

yrid

o[2,

3-b]

indo

l26

148-

68-5

183.

2no

npol

arpK

a6.

79±

0.30

c

14.8

7±

0.40

d

0.3

(TA

98)0

.02

(TA

100)

MeA

αC

2-A

min

o-3-

met

hyl-9

H-p

yrid

o[2,

3-b]

indo

l68

006-

83-7

197.

2no

npol

arpK

a7.

08±

0.30

c

15.2

5±

0.40

d

0.2

(TA

98)0

.12

(TA

100)

(Con

tinu

ed)



Tabl

e1–

Cont

inue

d.

Stru

ctur

eA

bbre

viat

ion/

Chem

ical

nam

eCA

SN

oM

olec

ular

wei

ght

(g/

mol

)Pr

oper

ties

aM

utag

enic

ity

(×10³r

ev/μ

g)b

β-C

arbo

lines

Har

man

1-m

ethy

l-9H

-pyr

ido[

4,3-

b]in

dole

486-

84-0

182.

2no

npol

arpK

a8.

62±

0.30

c

15.8

7±

0.40

d

co-m

utag

enic

Nor

harm

an9H

-pyr

ido[

4,3-

b]in

dole

244-

63-3

168.

2no

npol

arpK

a7.

85±

0.10

c

15.4

4±

0.30

d

co-m

utag

enic

γ-C

arbo

lines

Trp-

P-1

3-A

min

o-1,

4-di

met

hyl-5

H-p

yrid

o[4,

3-b]

indo

le62

450-

06-0

211.

3no

npol

arpK

a10

.88

±0.

10c

16.0

2±

0.40

d

39(T

A98

)1.7

(TA

100)

Trp-

P-2

3-A

min

o-1-

met

hyl-5

H-p

yrid

o[4,

3-b]

indo

le62

450-

07-1

197.

2no

npol

arpK

a10

.59

±0.

30c

15.5

9±

0.40

d

104.

2(T

A98

)1.8

(TA

100)

δ-C

arbo

lines

Glu

-P-1

2-A

min

o-6-

met

hyld

ipyr

ido[

1,2-

a:3’

2’-d

]imid

azol

e67

730-

11-4

198.

3no

npol

arpK

a6.

33±

0.30

c

49(T

A98

)3.2

(TA

100) (C

onti

nued

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Heterocyclic amines in cooked meat products . . .Ta

ble

1–Co

ntin

ued.

Stru

ctur

eA

bbre

viat

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Chem

ical

nam

eCA

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olec

ular

wei

ght

(g/

mol

)Pr

oper

ties

aM

utag

enic

ity

(×10³r

ev/μ

g)b

Glu

-P-2

2-A

min

o-di

pyrid

o[1,

2-a:

3’2’

-d]im

idaz

ole

6773

0-10

-3

184.

3no

npol

arpK

a5.

80±

0.30

c

1.9

(TA

98)1

.2(T

A10

0)

Oth

erpy

roly

ticH

AA

s

Phe-

P-1

2-A

min

o-5-

phen

ylpy

ridin

e33

421-

40-8

170.

2no

npol

arpK

a6.

32±

0.13

c

na

Orn

-P-1

4-A

min

o-6-

met

hyl-1

H-2

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0,10

b-te

traa

zaflu

oran

then

e78

859-

36-6

237.

3no

npol

arpK

a9.

52±

0.20

c

na

Cre-

P-1

4-A

min

o.1,

6-di

met

hyl-2

-met

hyla

min

o-1H

,6H

-pyr

rolo

-[3,4

-f]be

nzim

idaz

ole-

5,7-

dion

e13

3883

-91-

7

259.

3no

npol

arpK

a4.

83±

0.20

c

19(T

A98

)0.4

(TA

100)

Lys-

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1,2,

3,8-

Tetr

ahyd

ro-c

yclo

pent

a[c]

pyrid

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477-

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258.

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processes. Heterocyclic aromatic amines are similarly detected inrain water and cigarette smoke condensate (Xu and others 2010;Liu and others 2013). Individual HAAs were also detected in parti-cles from diesel exhaust fumes (Kataoka 1997). Over 25 mutagenicHAAs have been isolated and identified since 1977 (Alaejos andAfonso 2011) (Table 1). However, the concentrations detected infood vary widely, as will be discussed later.

The HAAs occurring most often in meat are PhIP, MeIQx,4,8-DiMeIQx, IQ, MeIQ, and AαC (Skog and others 1998a, b).The PhIP concentrations in most studies are between 1 and 70ng/g meat (Gross and Grueter 1992; Alaejos and Afonso 2011).Concentrations of MeIQx up to 23 ng/g can be detected (Skogand others 1995). As a rule, concentrations of up to 6 ng/g meat arefound (Skog and others 1998b). 4,8-DiMeIQx is mostly detectedonly in the lower concentration range of around 1 ng/g meat. Insome studies, IQ could not be detected at all (Skog and others1998b). Table 2 to 4 show an overview of the occurrence ofHAAs in beef patties, chicken breasts, and pork using differentpreparation methods.

While meat and meat products are frequently studied, fewerstudies were found about processed meat (Table 5). However, IQlevels of 3.8 to 10.5 ng/g were recorded in fried bacon (Johans-son and Jägerstad 1994). IQ and MeIQ could also be detected ingrilled sausages (Gerbl and others 2004). The HAA levels of ba-con prepared in different ways were investigated in a study (Sinhaand others 1998). Levels of 0.4 to 4.3 ng MeIQx/g and 0 to4.8 ng PhIP/g could be determined with pan-frying. MeIQxlevels of 0 to 4 ng/g with ovenroasting differed only a littlefrom frying. However, significantly higher PhIP levels of 1.4 to30.3 ng/g were detected for oven roasting (Sinha and others 1998).Further studies show that high HAA concentrations can be foundin bacon (Table 5). The use of various preparation methods affectsthe formation of HAAs differently for different meat products. Thegrill sausages investigated were pan-fried or grilled (Abdulkarimand Smith 1998). Along with different heating times, the heatingtemperature was also varied. With both cooking methods, alongwith norharman and harman, MeIQx and PhIP could also bedetected (Table 5). In offal products (beef liver, lamb kidney, andbeef tongue), which were thermally processed, HAAs were foundin concentrations only near the detection limit, except for norhar-man and harman. Both β-carbolines were found in concentrationsbelow 2 ng/g; only DMIP, MeIQx, and 4,8-DiMeIQx were de-tectable in cooked kidney and tongue up to 0.25 ng/g (Khan andothers 2009). The reason for the low contents of IQ-type HAAsmay be the lack of the precursor creatine (Harris and others 1997).The wide-ranging variation of precursors observed in studies withmeat from various animal species (Zimmerli and others 2001; Skogand Solyakov 2002; Sun and others 2010; Puangsombat and oth-ers 2012; Zaidi and others 2012), clearly affected the formation ofHAAs (Liao and others 2010, 2011b; Gibis and Weiss 2015).

Also the pH-value is known to influence the Maillard reactions(Cremer and Eichner 2000) and thereby also the formation ofHAAs. This was shown that the content of HAAs in pork increasedabout 22% on average (MeIQx – 33%; 4,8-DiMeIQx – 17%; andharman – 9%) in the PSE (pale, soft, and exudative) meat sampleswith lower pH values when grilled than the normal muscle at acore temperature of 95 °C (Polak and others 2009b). At the lowercore temperature of 70 °C, no remarkable difference in HAAformation could be observed between PSE meat and normal meat(Table 4). PSE is associated with pale color, low pH, and a highdrip loss causing by preslaughter stress and genetics of pigs witha fast post mortem glycolysis (Polak and others 2009b). In a study

investigating normal muscle meat of different animal species, themeasured pH values of the uncooked meat of the different animals(range of pH: 5.56 to 6.12) showed no significant linear correlationto the HAA levels, except for PhIP with a very weak correlation(r = 0.27, p < 0.05) (Gibis and Weiss 2015). Additionally, theduration of aging influenced the HAA concentrations for bothPSE and normal pork. Significantly higher levels were observedafter longer aging of pork (Polak and others 2009b) and beef (Polakand others 2009a).

Meat from horses showed threefold higher glucose levels thanbeef (Rossier 2003), which reduced the content of HAAs (Gibisand Weiss 2015). Similar results were observed in pork containinghigh and low levels of glucose (Olsson and others 2002). Contrar-ily, chicken had very low glucose levels, but a similar content ofcreatine, which increased, in particular, the concentration of PhIPby about a factor of 10 (Gibis and Weiss 2015). The same decreasein concentrations of PhIP, and less in levels of other HAAs, wasobserved in a model system by the addition of saccharides (Skogand Jägerstad 1990).

Formation of Heterocyclic Aromatic AminesFormation of imidazoquinolines and imidazoquinoxalines

The pyridines and pyrazines formed from hexoses and aminoacids, respectively, in the Maillard reaction via the Strecker degra-dation serve as building blocks for the IQ compounds. The reac-tion is depicted in Figure 1A. The creatine cyclizes to creatinineduring heating and reacts in an aldol reaction with the pyridineor pyrazine derivatives, respectively, to generate imidazoquinolineand imidazoquinoxaline (Skog and Jägerstad 1993). The aldehy-des arising, together with creatinine, also play an important rolein the formation of the imidazole rings of the polar HAAs. The2 parts can be joined to each other via a Strecker aldehyde to aSchiff base. The mechanism was confirmed for IQx, MeIQx, and4,8-DiMeIQx by using 14C-labeled glucose (Skog and Jägerstad2005).

Formation of PhIPPhenylalanine, reducing sugars and creatinine could be detected

in PhIP formation. It could be shown in a model trial with radioac-tively labeled carbon in the phenylalanine molecule that the phenylring was completely built into the PhIP molecule (Zöchling andMurkovic 2002). Further trials showed that creatinine forms apart of the imidazole ring. The authors were able to identifythe following reaction steps in PhIP formation in a model trial:First, phenylacetaldehyde is formed from phenylalanine via theStrecker degradation. The phenylacetaldehyde formed reacts inan aldol reaction with creatinine to form an intermediate prod-uct. In the subsequent condensation reaction, PhIP arose from thissubstance (Zöchling and Murkovic 2002). The mechanism of thereaction is shown in Figure 1B. The formation of formaldehydefrom phenylacetaldehyde and phenylalanine, and the combinationof both formaldehyde and ammonia in the generation of PhIPfrom phenylacetaldehyde and creatinine were reported in the re-action pathways that produce PhIP (Zamora and others 2014). Inthe presence of oxidized lipid, other amino acids competed withphenylalanine for the lipid, and amino acid degradation productswere formed, among which α-keto acids seemed to play a role inthese reactions (Zamora and others 2013b). However, unoxidizedlipids did not contribute to PhIP formation (Zamora and others2012).



Tabl

e2–

Occ

urre

nce

ofH

AA

sin

grou

ndbe

efpa

ttie

sus

ing

diff

eren

the

atin

gco

ndit

ions

.

Cook

ing

Cook

ing

tim

eTe

mpe

ratu

rePh

IPM

eIQ

x4,

8-D

i-MeI

Qx

Nor

harm

anH

arm

anO

ther

spr

oced

ure

(min

)(°

C)(n

g/g)

b(n

g/g)

b(n

g/g)

b(n

g/g)

c(n

g/g)

c(n

g/g)

bcRe

fere

nce

Frie

d4–

2015

0nd

–1.8

nd–0

.6nd

1.1

na(K

nize

and

othe

rs19

94)

190

nd–9

.80.

1–1.

30.

40.

15na

230

1.3–

320.

4–7.

3nd

1.6

naFr

ied

1219

84.

94.

31.

3na

naAα

C(2

1)(T

hiéb

aud

and

othe

rs19

95)

277

6816

4.5

Frie

d8

165

0.08

0.2

ndna

na(J

ohan

sson

and

othe

rs19

95b)

200

1.5

1.6

0.4

Frie

d5–

715

00.

01nd

ndna

na(S

kog

and

othe

rs19

95)

225

1.1

2.2

0.8

Frie

d–

–67

.516

.44.

5na

na(K

nize

and

othe

rs19

97a)

Gril

led

––

502

ndBa

rbec

ued

6+

620

02.

350.

27nd

1.87

0.61

Trp-

P-2

(1.7

)(A

bdul

karim

and

Smith

1998

)(1

5%fa

t)3.

5+

3.5

240

0.10

0.80

nd2.

170.

88Tr

p-P-

2(1

.5)

Frie

d3

+3

150

0.43

nd0.

310.

960.

31(1

5%fa

t)5

+5

190

0.57

0.84

2.0

0.83

7.5

+7.

523

00.

251.

005.

651.

7Fr

ied

12–2

017

50.

9–6.

20.

5–0.

80.

8–0.

9na

naIQ

(0.7

-1.3

),M

eIQ

(0.1

-0.3

)(B

alog

han

dot

hers

2000

)20

04.

0–25

.41.

5–4.

20.

9–4.

5IQ

(1.7

-4.4

),M

eIQ

(0.5

-2.1

)22

513

.3–1

.43.

5–5.

83.

0–4.

8IQ

(2.8

-5.3

),M

eIQ

(2-3

.5)

Frie

d–

–nd

–1.5

nd–1

.3nd

nana

(Zim

mer

lian

dot

hers

2001

)Fr

ied

323

00.

21.

0nd

2.1

1.5

(Jau

tzan

dot

hers

2008

)4.

523

00.

92.

00.

45.

03.

56

230

3.8

4.8

3.0

10.4

8.9

Frie

d/10

180

0.7

1.0

0.2

nana

dIQ

(0.1

/0.

05),

IFP

(0.1

/nd

)(N

iand

othe

rs20

08)

Ove

n-br

oile

d10

186

ndnd

ndIQ

[4,5

-b](

0.3/

nd),

IgQ

x(0

.5/

nd),

7-M

eIgQ

x(2

.4/

0.3)

,6,7

-DiM

eIgQ

x(0

.2/

0.05

),7,

9-D

iMeI

gQx

(0.7

/nd

)Fr

ied/

1518

92.

73.

00.

6na

nad

IQ(0

.1/

0.06

),IF

P(0

.6/

nd)

IQ[4

,5-b

](0.

3/0.

4),I

gQx

Ove

n-br

oile

d15

189

0.06

0.02

0.02

(1.5

/0.

03),

7-M

eIgQ

x(9

.5/

0.1)

,6,7

-DiM

eIgQ

x(0

.3/

nd),

7,9-

DiM

eIgQ

x(2

.2/

0.02

)Fr

ied/

2019

12.

93.

70.

7na

nad

IQ(0

.2/

0.1)

,IFP

(0.7

/0.

14)

IQ[4

,5-b

](0.

3/0.

15),

IgQ

xO

ven-

broi

led

2019

11.

230.

380.

1(1

.8/

0.3)

,7-M

eIgQ

x(1

1.7/

0.3)

,6,7

-DiM

eIgQ

x(0

.4/

nd),

7,9-

DiM

eIgQ

x(3

.0/

0.12

)Fr

ieda

4.5

230

2.6

4.9

1.8

13.5

21.4

(Gib

is20

09)

Frie

da2-

3̴19

00.

1–0.

20.

2–1.

7nd

–0.2

0.2–

0.9

0.7–

1.7

(Gib

isan

dW

eiss

2010

)

aFr

ied/

grill

edon

ado

uble

-pla

tegr

ill;b

ndno

tdet

ecte

d;c n

ano

tana

lyze

d;d

HA

Ang

/g

(frie

d/ov

en-b

roile

d).



Tabl

e3–

Occ

urre

nce

ofH

AA

sin

chic

ken

brea

st.

Cook

ing

proc

edur

eCo

okin

gti

me

(min

)

Cook

ing

tem

pera

ture

(°C)

PhIP

(ng/

g)c

MeI

Qx

(ng/

g)bc

4,8-

DiM

eIQ

x(n

g/g)

bcN

orha

rman

(ng/

g)b

Har

man

(ng/

g)c

Oth

ers

(ng/

g)Re

fere

nce

Pan-

frie

d14

–36

197–

221

12–7

01–

31–

4na

na(S

inha

and

othe

rs19

95)

Barb

ecue

d10

–43

177–

260

27–4

80nd

–9nd

–2na

naBo

iled

9–17

79–8

66–

150

nd-3

ndna

naPa

n-fr

ied

1617

50.

7na

nana

na(P

erss

onan

dot

hers

2002

)18

200

10.5

nana

nana

1222

529

.7na

nana

naPa

n-fr

ied

12–3

414

0–22

5<

0.1–

38.2

0.1–

1.8

0.1–

0.6

0.5–

6.9

0.3–

7.5

(Sol

yako

van

dSk

og20

02)

Roas

ted

24–4

017

5–24

0nd

ndnd

<0.

1–3.

3<

0.1–

1.7

Dee

p-fa

tfrie

d11

160

<0.

1a<

0.1a

<0.

1a0.

50.

3Bo

iled

3820

0nd

nd0.

1a<

0.1a

<0.

1aPa

n-fr

ied

14–3

619

7–21

18.

8–48

.50.

6–2.

30.

8–3.

6na

naIQ

(0.1

-0.2

),IQ

x(0.

1-0.

2),

IFP(

2.7-

15.9

),IQ

[4,5

-b](

a-0

.2),

IgQ

x(0.

3-0.

9),

(Nia

ndot

hers

2008

)

7-M

eIgQ

x(1.

5-8.

7),

6.7-

DiM

eIgQ

x(a

-0.1

),7.

9-D

iMeI

gQx(

0.2-

0.9)

,Aα

C(a

-0.1

),M

eAα

CaO

ven-

broi

led

9–17

179–

186

5.6–

72.0

0.1–

2.8

0.1–

2.0

nana

IQ(0

.2-0

.3),

IQxa

,IF

P(0.

4-11

.3),

IQ[4

,5-b

](a

-0.3

),Ig

Qx(

0.2-

0.4)

,7-

MeI

gQx(

1.0-

11.1

),6.

7-D

iMeI

gQx(

a-0

.2),

7.9-

DiM

eIgQ

x(0.

1-1.

8),

Aα

C(0.

2-9,

4),

MeA

αC(

a-1

.3)

Frie

dd5

220

2.4

2.1

0.11

0.07

0.09

(Gaš

perli

nan

dot

hers

2009

)G

rille

de18

220

0.3

0.2

0.13

0.11

0.05

Frie

dd4.

523

03.

80.

2nd

0.8

0.9

(Gib

is20

09)

Pan-

frie

d5

+5

180

18.3

1.8

1.1

1.4

2.8

IQ(1

.8),

Aα

C(0.

2),

MeA

αCa

(Lia

oan

dot

hers

2010

)

Dee

p-fa

tfrie

d10

180

2.2

0.8

0.4

5.4

12.3

Aα

C(0.

3),M

eAα

CaCh

arco

al-g

rille

d10

+10

200

31.1

1.2

3.6

32.2

31.7

Trp-

P-2(

3.6)

,Trp

-P-1

(1.5

),Aα

C(5.

6),M

eAα

C(1.

6)Ro

aste

d20

200

0.04

ndnd

3.1

0.7

Aα

Ca,M

eAα

Ca

aTr

aces

(�0.

05ng

/g)

.b

na,n

otan

alyz

ed.

c nd,

notd

etec

ted.

dFr

ied

ona

doub

le-p

late

grill

.e

Gril

led

onan

infr

ared

grill

.



Tabl

e4–

Occ

urre

nce

ofH

AA

sin

pork

.

Mat

eria

lCo

okin

gpr

oced

ure

Cook

ing

tim

e(m

in)

Cook

ing

tem

pera

ture

(°C)

PhIP

(ng/

g)a

MeI

Qx

(ng/

g)a

4,8-

DiM

eIQ

x(n

g/g)

aN

orha

rman

(ng/

g)b

Har

man

(ng/

g)a

bO

ther

s(n

g/g)

bRe

fere

nce

Pork

chop

Frie

d8–

9.5

150–

225

nd–4

.8nd

–2.6

nd–1

.1na

na(S

kog

and

othe

rs19

95)

Pork

chop

Frie

d5–

1517

5nd

nd–3

.8nd

nana

(Sin

haan

dot

hers

1998

)Po

rkpa

ttie

s(70

g)RN

− alle

leFr

ied

3+

320

01.

91.

90.

4na

na(O

lsso

nan

dot

hers

2002

)N

onRN

− alle

leFr

ied

0.2

1.5

0.2

nana

Pork

chop

sRN

− alle

lepH

5.32

Pan-

frie

d3

+3

160–

200

0.05

–0.1

0.1–

0.2

nd0.

6–1.

71.

1–0.

7IQ

x(0

.1–0

.2)

(Ols

son

and

othe

rs20

05)

Non

RN− a

llele

pH5.

58Pa

n-fr

ied

0.1–

3.3

0.1–

0.8

ndnd

ndIQ

x(n

d)Po

rkpa

ttie

sBo

iled

8–16

100

nd0.

4–1.

0nd

nana

(Shi

n20

05)

Broi

led

12–1

917

7–22

5nd

–2.7

1.2–

1.6

0.3–

0.7

nana

Pan-

Frie

d9–

2117

7–22

50.

3—10

–50.

6–5.

00.

3–1.

7na

naPo

rkst

eakc

Pan-

frie

d22

00.

8–3.

10.

6–4.

60.

2–1.

00.

2–0.

50.

9–0.

2(P

olak

and

othe

rs20

09b)

pH5.

48co

rete

mp

pH5.

6070

–95

0.8–

2.7

0.9–

3.1

0.2–

0.9

0.2–

0.5

0.9–

0.2

Pork

top

loin

Pan–

frie

d8

+8

204

1.8

1.1

1.2

nana

IQx

(nd)

(Pua

ngso

mba

tand

othe

rs20

12)

Bake

d70

2.2

0.2

0.9

nana

IQx

(nd)

Pork

patt

yFr

ied

(50

g)5

180

18.4

3.2

0.7

nana

(Zha

ngan

dot

hers

2013

)Po

rkm

eat-

ball

5.3

1.0

0.3

nana

Pork

strip

5.4

1.0

0.3

nana

Pork

loin

Pan-

frie

d20

4co

rete

mp

7713

.17.

61.

6na

na(V

angn

aian

dot

hers

2014

)

Pork

patt

ies(

80g)

pH5.

56G

rille

dd2.

722

02.

31.

10.

51.

10.

5(G

ibis

and

Wei

ss20

15)

ana

nota

naly

sed,

bnd

notd

etec

ted.

c M.l

ongi

ssim

usdo

rsi

dfr

ied

ona

doub

le-p

late

grill



Tabl

e5–

Com

pari

son

ofth

eco

nten

tof

the

mos

tco

mm

onH

AA

sin

cook

edba

con

and

proc

esse

dm

eat.

Hea

ting

cond

itio

nH

AA

a

Prep

arat

ion

met

hod

Tim

e(m

in)

Tem

p.(°

C)M

eIQ

x(n

g/g)

4,8-

DiM

eIQ

x(n

g/g)

PhIP

(ng/

g)N

orha

rman

(ng/

g)H

arm

an(n

g/g)

Refe

renc

e

Baco

nfr

ied

12–1

617

00.

9–27

0.5–

2.4

nd–5

2nd

–22

nd–3

0(G

ross

and

othe

rs19

93)

Baco

nm

icro

-wav

ed(6

00W

)3

0.1

ndnd

3.3

ndBa

con

pan-

frie

d5

150

2.8

3.4

0.2

nana

(Joh

anss

onan

dJä

gers

tad

1994

)Ba

con

pan-

frie

d4–

1617

6–17

70.

4–4.

3<

0.2

<0.

2–4.

8na

na(S

inha

and

othe

rs19

98)

Baco

nov

en-b

roile

d4–

717

5–18

50.

2–4

<0.

21.

4–30

.3na

naBa

con

mic

ro-w

aved

1.8–

3.3

<0.

2–1.

5<

0.2

<0.

2–3.

1na

naBa

con

pan-

frie

d1.

523

08.

14.

528

.4na

na(G

uyan

dot

hers

2000

)Ba

con

grill

ed1.

523

01.

60.

95.

0na

naBa

con

pan-

frie

d16

.117

63.

00.

74.

9na

na(N

iand

othe

rs20

08)

Baco

nov

en-b

roile

d7.

517

52.

65.

215

.9na

naBa

con

pan-

frie

d3

204

4.0

3.6

6.9

nana

(Pua

ngso

mba

tand

othe

rs20

12)

Baco

npa

n-fr

ied

3–6

160

1.5–

4.9

nd0.

1–1.

15–

14.1

0.4–

1.7

(Gib

isan

dot

hers

2015

)2–

321

02.

4–5.

6nd

1.3–

2.6

13.7

–19.

91.

3–1.

6Fa

lun

saus

age

frie

d5

160

0.6

ndnd

nana

(Joh

anss

onan

dJä

gers

tad

1994

)Sa

usag

efr

ied

616

00.

70.

20.

1na

naH

ampa

n-fr

ied

5–19

175

<0.

2–1.

8<

0.2

<0.

2–0.

3na

na(K

nize

and

othe

rs19

97b)

Hot

dogs

pan-

frie

d4–

1817

5–17

7nd

ndnd

nana

(Sin

haan

dot

hers

1998

)H

otdo

gsov

en-b

roile

d3–

1018

0–18

5nd

ndnd

nana

Hot

dogs

grill

/ba

rbec

ued

5–15

232–

252

ndnd

ndna

naPo

rksa

usag

efr

ied

6–15

150–

230

nd–0

.7nd

nd–1

.1nd

–0.8

nd–3

.1(A

bdul

karim

and

Smith

1998

)Po

rksa

usag

eba

rbec

ued

12/

720

0/24

00.

35/

0.8

nd0.

1/1.

36.

1/1.

160.

57/

4.2

Saus

age

frie

d9

175–

200

<0.

2<

0.5

<0.

10.

30.

3(B

usqu

etsa

ndot

hers

2004

)Po

rksa

usag

epa

ttie

sfrie

d21

179

5.1

0.7

0.2

<0.

3<

0.3

(Nia

ndot

hers

2008

)Pi

zza

topp

ing

sala

mio

ven-

bake

d12

–20

230

nd–2

.6nd

nd–0

.410

7.4–

186.

111

.4–2

4.7

(Gib

isan

dW

eiss

2013

)10

–12

250

nd–0

.2nd

0.1–

0.3

143.

2–14

6.1

15.3

–21.

0Pi

zza

topp

ing

ham

oven

-bak

ed12

–20

230

0.2–

3.1

0.5–

2.1

0.2–

0.8

4.5–

10.3

2.5–

4.8

10–1

225

00.

1–0.

20.

5–0.

60.

3–0.

55.

6–7.

04.

3–4.

9a

na,n

otan

alyz

ed;n

d,no

tdet

ecte

d.



Tabl

e6–

Effe

ctof

diff

eren

tan

tiox

idan

tin

gred

ient

son

the

form

atio

nof

HA

As

infr

ied

orgr

illed

beef

prod

ucts

,inh

ibit

ion

ofH

AA

form

atio

n(%

)and

incr

ease

ofH

AA

leve

lsco

mpa

red

toth

eco

ntro

lsw

itho

utan

tiox

idan

tco

mpo

nent

sas

indi

cate

dby

+;na

indi

cate

sno

tan

alyz

ed;n

din

dica

tes

not

dete

cted

.

Prod

uct

Ingr

edie

ntCo

ncen

trat

ion

Cook

ing

tim

e(m

in)

Cook

ing

tem

p.(°

C)Ph

IP(%

)M

eIQ

x(%

)

4,8-

DiM

eIQ

x(%

)N

orha

rman

(%)

Har

man

(%)

Oth

ers

(%)

Refe

renc

e

Beef

patt

yCh

erry

tissu

e11

.5%

8+

817

093

6281

nana

IQ(7

2),M

eIQ

(50)

(Brit

tand

othe

rs19

98)

Beef

stea

kRo

sem

ary

Spre

ada

2018

075

3839

nana

IQ(7

2),M

eIQ

(64)

(Mur

kovi

can

dot

hers

1998

)G

arlic

Spre

ada

5471

78na

naIQ

(32)

,MeI

Q(4

0)Sa

geSp

read

a10

040

100

nana

IQ(1

00),

MeI

Q(7

7)Th

yme

Spre

ada

7561

100

nana

IQ(7

4),M

eIQ

(61)

Beef

patt

yO

leor

esin

rose

mar

y1%

10+

1022

544

3077

nana

IQ(7

2),M

eIQ

(87)

(Bal

ogh

and

othe

rs20

00)

10%

4512

68na

naIQ

(72)

,MeI

Q(7

2)V

itam

inE

1%69

4879

nana

IQ(8

6),M

eIQ

(79)

10%

7226

71na

naIQ

(88)

,MeI

Q(6

4)Be

efpa

tty

(100

g;15

.4%

fat)

Min

ced

garli

c4.

5%10

+10

225

3413

24na

na(S

hin

and

othe

rs20

02a)

18.2

%,

7162

62na

naSu

lfurb

Com

p.0.

17m

M51

2846

nana

0.67

mM

8266

81na

na1.

01m

M90

7081

nana

Beef

patt

yV

irgin

oliv

eoi

lfre

sh40

g5

+5

200

605

nd50

40(P

erss

onan

dot

hers

2003

a)(9

0g)

Stor

edfo

r1ye

ar75

65nd

800

Beef

patt

yG

rape

-see

d0.

5%10

+10

210

126

100

48+3

493

IQ(1

00),

MeI

Q(1

7),

Aα

AC(

29)

(Ahn

and

Gru

en,2

005b

)

1%25

6410

064

+694

9IQ

(100

),M

eIQ

(34)

,Aα

AC(

43)

Pine

bark

0.5%

3747

100

5528

IQ(1

00),

MeI

Q(1

00),

Aα

AC(

+11)

1%36

6210

061

32IQ

(100

),M

eIQ

(100

),Aα

AC(

29)

Ole

ores

inro

sem

ary

0.5%

396

2449

23IQ

(100

),M

eIQ

(26)

,Aα

AC(

100)

1%58

2310

010

055

IQ(1

00),

MeI

Q(1

00),

Aα

AC(

100

BHA

/BH

T0.

02%

12+3

1552

27IQ

(100

),M

eIQ

(4),

Aα

AC(

17)

Beef

patt

yG

rape

-see

d0.

1%6

+6

210

7267

66na

na(C

heng

and

othe

rs20

07)

App

le0.

1%69

5963

nana

Elde

rber

ry0.

1%45

619

nana

Pine

appl

e0.

1%13

2718

nana

Beef

patt

yCa

rvac

rol

1%20

0co

rete

mp

7078

72nd

nana

MeI

Q(5

8)(F

riedm

anan

dot

hers

2009

)

Beef

patt

y(1

00g)

Min

ced

garli

c4.

8%5

+5

220

1651

nd6

6Tr

p-P-

1(15

),Tr

p-P-

2,G

lu-P

-1,&

Glu

-P-2

(100

),Aα

AC

(+39

)

(Jun

gan

dot

hers

2010

)

13%

8310

0nd

9688

Trp-

P-1(

100)

,Trp

-P-2

,G

lu-P

-1,&

Glu

-P-2

(100

),Aα

AC

(100

)Be

efpa

tty

(70

g)H

ibis

cusm

arin

adec

0.2%

2.7

230

1836

nd+1

+2(G

ibis

and

Wei

ss20

10)

0.8%

6456

nd+6

6+3

3

(Con

tinu

ed)



Tabl

e6–

Cont

inue

d.

Prod

uct

Ingr

edie

ntCo

ncen

trat

ion

Cook

ing

tim

e(m

in)

Cook

ing

tem

p.(°

C)Ph

IP(%

)M

eIQ

x(%

)

4,8-

DiM

eIQ

x(%

)N

orha

rman

(%)

Har

man

(%)

Oth

ers

(%)

Refe

renc

e

Beef

patt

yG

alan

gal

0.2%

5+

520

419

18na

nana

(Pua

ngso

mba

tand

othe

rs20

11)

(100

g)Fi

nger

root

0.2%

3731

nana

naTu

rmer

ic0.

2%38

41na

nana

Cum

in0.

2%6

1na

nana

Coria

nder

seed

s0.

2%6

3na

nana

Rose

mar

y0.

2%36

50na

nana

Beef

patt

y(7

0g,

froz

en)

Gra

pe-s

eedd

0.2%

2.7

230

5012

nd+3

+6(G

ibis

and

Wei

ss20

12)

0.8%

7193

nd+2

5+5

5Ro

sem

ary

extr

acte

0.12

%35

16nd

+34

+55

1.5%

9154

nd+5

03

Beef

patt

y(3

0g,

6.2×

1.2

cm)

Asc

orbi

cac

id0.

02m

M3

+3

200

1917

14na

na(W

ong

and

othe

rs20

12)

Nia

cin

1919

15na

naPy

rido

xam

ine

4342

38na

na

Beef

stea

k(1

.4cm

thic

k)

Beer

50%

3+

318

050

>99

>98

nana

IQ(>

97)

(Vie

gasa

ndot

hers

2012

)Be

er+

spic

esf

91>

7664

nana

IQ(>

97)

Win

e+1

>76

70na

naIQ

(>97

)W

ine

+sp

ices

f32

3364

nana

IQ(7

2)D

Ag

Beer

+sp

ices

f61

52>

98na

naIQ

(>97

)D

Ag

Win

e+

spic

esf

7751

>98

nana

IQ(>

97)

Beef

patt

y(6

2.5

g,10

mm

thic

k)

Soy

leci

thin

h1%

L2.

722

0+2

047

nd+2

+21

(Nat

ale

and

othe

rs20

14)

5%L

+90

26nd

+8+1

6So

yle

cith

inh+

GSE

i1%

L+

0.1%

GSE

5047

nd1

+24

5%L+

0.1%

GSE

7037

nd55

471%

L+

0.2%

GSE

1042

nd2

+21

5%L+

0.2%

GSE

+40

26nd

+20

+24

Pure

GSE

i0.

1%50

37nd

313

0.2%

2032

nd+2

3+5

aSp

ices

spre

adon

surf

ace.

bO

rgan

osul

furc

ompo

unds

:dia

llyld

isul

fide,

dipr

opyl

disu

lfide

,dia

llyls

ulfit

e,al

lylm

ethy

lsul

fide,

ally

lmer

capt

an,c

yste

ine,

cyst

eine

.c P

lant

extr

acts

,.5

gof

mar

inad

eus

edon

each

side

ofth

epa

tty.

dG

rape

-see

dor

Hib

iscu

sext

ract

inw

ater

-insu

nflow

eroi

lem

ulsi

on(m

arin

ade)

(em

ulsi

fierE

472c

:citr

icac

ides

ters

ofm

ono

and

digl

ycer

ides

);1.

5g

ofm

arin

ade

used

onea

chsi

de.

eRo

sem

ary

extr

acti

nsu

nflow

eroi

lmar

inad

e;1.

5g

ofm

arin

ade

used

onea

chsi

de.

f Mar

inad

eco

ntai

ning

ging

er(2

.8%

),ga

rlic

(2.9

%),

rose

mar

y(0

.4%

),th

yme

(0.2

5%

),an

dre

dch

ilipe

pper

(0.1

%,w

/v)

.g

DA

,dea

lcoh

oliz

edw

ine

orbe

er.

hSo

yle

cith

inco

ntai

ning

0.18

%D

L-α

-toc

ophe

rols

hom

ogen

ized

at22

500

psit

olip

osom

es(L

)use

das

mar

inad

e(1

0g,

mar

inat

ing

time

3h

at8°C

)com

pare

dto

cont

rol(

rape

seed

oilr

efine

d).

i Pur

eor

inlip

osom

esin

corp

orat

edG

SE(g

rape

-see

dex

trac

t).



Figure 1–Formation of heterocyclic amines: (A) mechanism of thermophilic HAAs with creatine, sugar, and amino acids (modified) (Jägerstad andothers 1983), (B) mechanism of formation of PhIP without sugar (modified) (Zöchling and Murkovic 2002), and (C) formation of norharman asβ-carboline (modified) (Roenner and others 2000).

Formation of β-carbolineThe reaction of the formation of β-carboline norharman is

shown in Figure 1C (Roenner and others 2000). A similarreaction is postulated for both β-carbolines (norharman and har-man). Both substances have no free amino group, which causes nomutagenicity in the Ames test (Pfau and Skog 2004). In compari-son to other carbolines (pyrolytic HAAs), the reaction of norhar-man and harman could be formed at lower temperatures and theyoccurred in cooked meat, fish, and meat extract (Ziegenhagen

and others 1999) as well as in processed meat, particularly suchpizza toppings as salami and cooked ham (Gibis and Weiss 2013).Tryptophan, in particular, was found as a precursor and glucoseenhanced the formation (Pfau and Skog 2004). Under the samecondition with an excess of tryptophan, harman and norharmanwere found at levels 70 and 20 times higher, respectively (Skog andothers 2000). The other carbolines are normally formed as typicalpyrolysis products of amino acids above a temperature of 300 °C(Jägerstad and others 1998).



Precursors of Heterocyclic Aromatic AminesCreatine and creatinine

Creatine, as a nonproteinogenic amino acid, occurs in the formof creatine phosphate in the muscle of vertebrates and serves as anenergy store. Heating transforms it into the physiologically inertcyclization product creatinine (Murkovic and others 1999). Theresulting creatinine is needed to form the imidazole ring. If nocreatinine is available, no imidazoquinoline and imidazoquinox-aline are formed. By contrast, the formation of nonpolar HAAsis independent of the presence of creatinine (Murkovic 2004b).The longer the heating time, the more creatine is converted tocreatinine. The higher the addition of creatine/creatinine duringthe heat treatment of meat and fish, the higher is the mutagenicityof the products (Felton and Knize 1991). A promoting indicationthat creatine is significantly involved in the formation of thermicHAAs is that there are only small amounts of creatine in protein-rich foods, such as cheese, beans, liver, and shrimp. Only very smallamounts of mutagenic activity could be detected in these productsafter preparation (Cheng and others 2006; Chen and others 1990).The molar ratios of total creatine/glucose was found from 0.89 upto 9.84 for beef, pork, mutton, chicken, duck, and goose (Liao andothers 2011b) and 1.2 up to 12 for veal, beef, pork, lamb, horse,turkey, and ostrich, respectively, except for vension and chickenwith ratios up to 60.7 (Gibis and Weiss 2015) in meat from dif-ferent animals. Trials with a model system showed that creatinineforms a part of the PhIP molecule (Zöchling and Murkovic 2002).It was shown in a further trial that beef with a high creatine level(1.5 mg/g) demonstrated a higher mutagenic activity than beefwith a low creatine level (Jackson and others 1994).

Free amino acidsFree amino acids are, similar to creatine, necessary for the

formation of HAAs. However, several different amino acids canform the same mutagens, for example, threonine, glycine, lysine,alanine, and serine all lead to MeIQx formation (Jägerstad andSkog 1991). Several amino acids were heated in the presence ofcreatinine and glucose in model trials and all the amino acidsshowed mutagenic activity (Johansson and others 1995a). Otherauthors showed that some amino acids had less mutagenic activitywith dry heating. According to this study, the amino acids serineand threonine had the highest mutagenic activity (Overvik andothers 1989). Which mutagens are formed depends on the aminoacids present. PhIP is formed from the amino acid phenylalanine(Manabe and others 1992, 1993). Other authors determined,using 13C NMR spectrometry, that a 13C-labeled phenylalaninemolecule is built into the structure of the PhIP molecule(Murkovic and others 1999). In model studies, phenylalanine, as aknown precursor for PhIP, is highest in the chicken model system,and the compound is also formed from tyrosine and isoleucine,which are similarly highest in chicken (Skog and others 1998b).However, no correlation between the amount of phenylalanineand the levels of PhIP was observed in pork (Olsson and others2002). MeIQx, by contrast, arose from the amino acids glycine,threonine, alanine and lysine (Johansson and Jägerstad 1996;Johansson and others 1995a). In the model, the amino acidsthreonine, alanine, and lysine took part in the formation of4,8-DiMeIQx (Johansson and others 1995a). The amino acidsglycine, alanine, and lysine were responsible for the formationof 7,8-DiMeIQx. With the exception of L-cysteine, whichreduced the mutagenicity in the Ames test, all amino acidsincreased the mutagenic activity (Lee and others 1994). When4-oxo-2-nonenal, a compound of oxidization of fat, was present,only the addition of methionine, glycine, and serine significantly

increased the amount of PhIP formed (p < 0.05), whereascysteine, lysine, tryptophan, histidine, tyrosine, and alaninereduced (p < 0.05) PhIP significantly (Zamora and others 2013b).The generation of PhIP is also associated with the capacityof 4-oxo-2-nonenal to constitute the Strecker degradation ofphenylalanine to phenylacetaldehyde (Zamora and others 2013a).Amino acids also formed HAA amino acid adducts, for example,the PhIP adduct with glycine was formed easily within 5 min byheating at 200 °C, which is probably based on the dehydrationcondensation of the amino group of PhIP and carboxyl groupof glycine (Kataoka and others 2012). The content of free aminoacids as the sum of all encoded and nonencoded amino acids is nothighly correlated with the formation of HAAs in grilled beef; butderivatives of amino acid, such as creatinine, and the amino acidsalanine, phenylalanine, and lysine are related to the formation ofaminoimidazoarenes and PhIP, while the other amino acids do notparticipate in the formation of HAAs (Szterk and Waszkiewicz-Robak 2014). Similar findings were observed in pork, beef, andchicken, only lysine, tyrosine, phenylalanine, proline, isoleucine,and aspartic acid showed significantly positive correlations to levelsof PhIP, while no significant correlation between any amino acidand contents of other HAAs was found (Gibis and Weiss 2015).

CarbohydratesThe presence of reducing sugars plays an important role in the

formation of HAAs. Along with creatine, creatinine, and aminoacids, reducing sugars such as glucose, fructose, ribose, erythrose,and lactose are also necessary for the formation of these muta-gens (Skog and Jägerstad 1993). Glucose was necessary for form-ing mutagenic activity in the Ames test both in aqueous modelsystems and with the heating of meat samples (Skog and others1992). The sugars occurring naturally in meat are present in ap-proximately half the molar quantity of creatine and amino acids.If the sugar concentration is increased beyond the naturally oc-curring ratio, a reduction is observed in HAA formation (Skogand Jägerstad 1990). The same was observed for glucose, fructose,sucrose, and lactose as for sugar, however, the monosaccharidesshowed the most distinct inhibitory effects (Skog and Jägerstad1990). Some authors found higher ratios of total creatine/glucosewith values from 0.89 up to 9.84 (Liao and others 2011b) andup to 60.7, respectively, (Gibis and Weiss 2015) for animals ofdifferent species. These ratios of the precursors to each other alsoplay a key role in the formation of HAAs. Higher levels werefound for MeIQx by adding lactose in model systems (molecularratio lactose/amino acids/creatinine – 2:1:1) with 5% water con-tent (Dennis and others 2015). The inhibition of the formationof the mutagenic compounds by an excess of sugars is proposedto be an effect of Maillard reaction products, which may blockthe formation of imidazoquinoxalines by attacking creatine (Skogand Jägerstad 1990). Therefore, the reason is due to the formationof Maillard products which changes the reaction path and resultsin a reduction in HAA concentration (Shin and others 2002c). Asignificant reduction in mutagenicity and in the content of MeIQ,PhIP, DiMeIQx, IQ, IQx, and norharman was attained in grilledchicken that was marinated with honey containing glucose andfructose in the same ratio and a small amount of sucrose (Hasnoland others 2014; Shin and Ustunol 2004). It was also observed in astudy with pork, from pigs as carriers of the RN allele, containingincreased concentrations of residual glycogen that this fact causedabout 90% lower levels of PhIP and 50% lower of total mutagenicHAAs in cooked meat compared with cooked meat from normalpigs (Olsson and others 2002).



Different oligosaccharides, such as fructooligosaccharide, galac-tooligosaccharide, isomaltooligosaccharide, or inulin, reduced theHAA formation in cooked meat products (Shin and others 2003,2004; Persson and others 2004) . The addition of oligosaccharidesaffected the mass transfer of the precursors to the surface, whichresults in higher water-holding capacity as well as thereby a lowerweight loss of cooked products and reduction of mass transfers ofprecursors (Persson and others 2004).

Physical FactorsHeating time and heating temperature

The formation of HAAs depends on the heating time and theheating temperature depending on the used cooking method. Theheating temperature has the largest effect on HAA formation.The formation of mutagenic substances begins at around 125 °C(Jägerstad and others 1998; Skog and others 1998b). Howeveraccording to Ahn and Gruen (2005a), few or no HAAs were de-tected at temperatures (160 °C) and a heating time of 15 min inground beef as a model systems with glass test tubes. Only afterincreasing the heating temperature and time, the HAA concen-tration rose. At 180, 200, and 220 °C, significant concentrationsof polar HAAs (IQ, MeIQ, MeIQx, 4,8-DiMeIQx, and PhIP)and nonpolar HAAs (harman, norharman, and AαC) were deter-mined after 20 min. By contrast, neither MeIQ nor 4,8-DiMeIQxcould be detected at 180 °C and a cooking time of 10 min. TheMeIQ concentrations detected were under 2 ng/g at all 3 tem-peratures (Ahn and Gruen 2005a). The HAA concentrations inthe trials performed increased only after about 5 min, but thendisproportionally. This observation could be explained by the sur-face temperature being under 100 °C in the first few minutes ofthe heating process. The temperature range that is required forthe formation of the mutagenic substances was only reached afterabout 5 min (Ahn and Gruen 2005a).

It could be shown in further experiments (Bordas and others2004) that the concentration of HAAs was increased with an in-crease in the temperature and an extension of the cooking time. A4,8-DiMeIQx concentration of 1.2 ng/g was detected in an aque-ous model system with meat flavor extract (bouillon, extract/water1:2) after heating for 1 h at 175 °C in an oven. With the same tem-perature and a doubling of the heating time, a threefold quantityof 4,8-DiMeIQx was detected. In this meat flavor model system indry conditions, changing the heating times and temperatures from100 °C for 1 h to 2 h, 150 °C for 30 min to 1 h, and 200 °C for10 min up to 30 min, respectively, made the effect of temperatureand time on HAA formation clear. While a MeIQx concentrationof 3.5 ng/g was found at 100 °C for both times, at 150 °C, therewas a concentration of around 4 ng/g for both heating times. Af-ter increasing to 200 °C, the concentration increased from 3.7 to10.7 ng/g for the longer heating time (Bordas and others 2004).A significant increase of PhIP concentration was already observedat 100 °C after 2 h compared to a heating time of 1 h (Bordas andothers 2004).

The results showed that HAA formation depends on temper-ature, time, and cooking methods. Further studies are shown inTable 2 to 4 concerning cooked meat samples (beef patties, chickenbreasts, and pork) under different cooking methods and at variousheating conditions.

Heat and mass transfer in cooked meat productsThe objectives of cooking meat products are to warrant mi-

crobial safety and to produce flavor in meat. Some chemical and

physical alterations take place during the cooking of meat, such asprotein denaturation, melting of fat, water evaporation, changesin texture and shape, water transport of solutes, and formationof crust on the surface (Kondjoyan and others 2014). The de-naturation of meat proteins during heating results in shrinkage,hardening, and the release of juice. First, the denaturation of themyosin takes place at approximately 53 to 58 °C, second, colla-gen shrinks and denatures at approximately 70 to 74 °C similaras sarcoplasmic proteins, and third actin denaturation at about 80to 82 °C results in a decrease in water-holding capacity with therelease of serum (Bertram and others 2006). Free amino acids andsugars in the outer parts of the meat react via the Maillard reac-tion during frying and form a variety of reaction products, whichare important for the color and flavor of cooked meat; aminoacids containing sulfur are known as precursors for the final flavor(Shahidi and others 2014).

The effect of temperature and time on HAA formation wasdescribed in the previous section. However, the cooking methodused also has a big effect on the formation of mutagenic activ-ity. Authors found in different studies that no amounts of HAAsor mutagenicity occurred with gentle cooking methods, such asstewing, steaming, and boiling as temperatures are below 120°C (Persson and others 2002; Joshi and others 2015) (Table 2and 3). With dry-heating methods, such as frying in a pan, sig-nificantly higher mutagen levels occur than with oven-roasting(Table 2 to 4). In addition to the temperatures and heating time,the methods differed between the type of heat transfer, such asconvection, conduction, or radiation, and the surrounding me-dia, such as metal, water, fat, or air, which result in different heattransfer coefficients (Houšová and Topinka 1985; Pan and Singh2002; Zorrilla and Singh 2003; Kondjoyan and others 2013). Itcould be shown in model trials that the preparation of hamburg-ers in a pan led to a much higher mutagenic activity and HAAconcentrations than oven-roasting. A comparison of the resultsof preparation in a deep-fat fryer, a convection oven, and on acontact grill showed clear differences (Persson and others 2002)(Table 2 and 3). MeIQx levels after preparation on a contact grillare around 10 times higher than with the other methods. Withpreparation in a deep-fat fryer or a convection oven, lower lev-els of HAAs were formed. The differences are explained by thesignificantly better heat transfer in methods with direct contact,such as grilling or frying in a pan (heat transfer coefficient of pan150, air 40 Wm2/K, and up to 10000 Wm2/K for boiling wa-ter), than in methods without direct contact (air convection 30to 40 Wm2/K) (Pan and Singh 2002; Sprague and Colvin 2011).There is an indirect heat application in ovenroasting as the heathas to be transferred by convection. There is also an indirect heattransfer using microwave methods, as the energy here is trans-ferred in the form of radiation (Haskaraca and others 2014; Singhand Heldman 2014). That is the reason why preparations of meatproducts using a microwave pretreatment result in very low HAAconcentrations compared to only charcoal-grilled or deep-friedchicken or beef samples (Felton and others 1994; Jinap and oth-ers 2013). However, Barrington and others (1990) showed a highmutagenic activity in 1 beef steak sample which were microwave-cooked for 5 to 7 min per side in contrast to lower times. Thistreatment most probably resulted in high water loss. The high-est HAA levels occurred when frying on a contact grill (Panand others 2000). The reason for this is the direct contact withthe heating medium, which results in a higher conductive heattransfer.



Kinetics of HAA formationThe kinetics of HAA formation was studied by using model

systems prepared with precursors, such as creatinine, glucose,dipeptides, and free amino acids, analogously to levels in beef(Arvidsson and others 1997) and beef juices as a model system,which were obtained from roasted beef (Arvidsson and others1999). The HAAs are stable at ambient temperature, but they aredisposed to degradation at higher temperatures (Jackson and Har-graves 1995; Arvidsson and others 1997). Degradation occurred at100 °C in solutions of standard HAAs and increased significantlywith temperatures at 200 to 225 °C. In this connection, PhIP wasfound to be the most disposed to degradation, followed by MeIQx,4,8-DiMeIQx, and IQx (Chiu and Chen 2000). However, degra-dation in meat juice systems or meat was different because theformation can balance more due to various parameters, such as heattransfer, mass transfer, vaporization of water, and crust formation,which complicate the kinetics calculations. Besides the generationreactions, the kinetics of HAAs also included a subsequent degra-dation of HAA compounds. The formation of HAAs ([HAA]levels in ng/g, t for time) is supposed to follow a first-order reac-tion equation with a rate constant given by the Arrhenius equation(Arvidsson and others 1997, 1999; Tran and others 2002):

∂ [HAA]

∂ t= A · e (− EaRT ) (1)

k = kb Th

· e ( �SR ) · e (− �HRT ), (2)

where Ea is the activation energy, R is the gas constant (8.3145J/(mol·K)), and A is the unknown exponential prefactor. Forthe reaction mechanism, the modulation of Eq. 1 was used inthe Eyring Eq. 2 for the determination of the temperature-dependence (T in K) of rate constant of the formation wherek is the rate constant, kb is the Boltzmann constant (1.381 × 10−23J/K), h is the Planck constant (6.626 × 10−34 J s), and �H isthe activation enthalpy (Arvidsson and others 1997, 1999). Theactivation entropy �S calculated showed that the rate-limitingstep for the formation of PhIP follows rather a monomolecularreaction, whereas for all the IQx-type compounds the formationprobably follows a bimolecular reaction of pseudo-first order (1 ofthe 2 reactants being in large excess) for MeIQx, 4,8-DiMeIQx,and IQx (Jägerstad and others 1998). The limiting step of the ratecould be the reaction between creatinine, aldehyde, and pyrazine(Arvidsson and others 1997, 1999). Using numerical methodswith different mathematical and computational modeling of pan-frying can show the predictive values of the transport of waterand the temperature distribution in patties, as well as the asso-ciated formation of HAAs, for turning once and multi-turning(Sprague and Colvin 2011). The modeling of the formation ofHAAs in slices of beef (musculus longissimus thoracis and semimem-branosus) subjected to jets of hot air depicted the influence ofextreme dehydration (low water activity) obtained, which slowedthe formation of IQx, MeIQx, and, particularly, 4,8-DiMeIQxcompared with superheated steam treatments. By contrast, a re-verse effect was found for PhIP levels, which increased 1.4- to5.5-fold (Kondjoyan and others 2010a; 2010b). In this connec-tion, the knowledge is important that many reactions favoringat various environmental conditions can simultaneously proceed.In model systems with high temperature around 225 °C or longcooking times, formation and degradation of the HAA in partic-ular PhIP were reported (Chiu and Chen 2000; Arvidsson and

others 1997). Additionally there is known from model studies thatdry conditions (freeze dried meat juice) resulted in an increasedformation of PhIP, DMIP, TMIP, and IFP, but in the wet system(meat juice), MeIQx and 4,8 DiMeIQx was favored (Borgen andothers 2001).

Chemical FactorsFat content and lipid oxidation products

No clear statement could be made on the role of fat in thedevelopment of mutagenic activity. The fat content of the productsaffected the formation of mutagenic substances, but it is not clearwhether this was due to physical or chemical influences (Johanssonand Jägerstad 1993; Hwang and Ngadi 2002).

Moreover, the precursors being necessary for HAA formation,such as creatine/creatinine, free amino acids, and carbohydrates,are present almost exclusively in lean meat, and a higher fat con-tent results in a dilution of the precursors (Knize and others 1985)leading to a reduction of the mutagenic activity in the Ames test(Chen and others 1990). However, it has also been shown thata higher fat content in meat results in a shorter time needed toreach a fixed meat surface temperature due to a more effectiveheat transfer and thereby an increased formation (Abdulkarim andSmith 1998). Furthermore, if the fat is oxidized the HAA forma-tion is enhanced that was shown in different studies (Johansson andJägerstad 1993; Zamora and others 2012). Nonetheless, many sci-entists agree that there is an optimal fat content for the formationof the highest possible HAA concentration in a product.

Regarding beef patties, HAAs such as MeIQx, PhIP, norhar-man, harman, and Trp-P-2 were found at higher levels with about5% fat than those with 15% fat after frying at 150, 170, and190 °C (Abdulkarim and Smith 1998). In beef patties with 15%and 30% fat content, which were cooked to a core temperature of100 °C on a propane grill using a cooking time from 10 up to26 min (weight loss from 46% to 55%), the low-fat patties had thehigher levels of PhIP, but lower levels of AαC than the high-fatpatties (Knize and others 1997c).

Lipids had increased the yield of pyrazines and Strecker aldehy-des in model trials (Arnoldi and others 1990). A possible reason forthis is fat oxidation. Oxidized fats led to radicals which supportedthe formation of HAAs by this oxidation (Johansson and Jägerstad1993). The content of HAAs in meat, including pan residue fry-ing with different frying fats, or oils (butter, margarine, margarinefat phase, liquid margarine, rapeseed oil, and sunflower seed oil),was significantly lower after frying in sunflower seed oil or mar-garine than after frying with the other fats (Johansson and others1995b). The variations in generation of MeIQx and DiMeIQxcould be stated with regard to the concentrations of antioxidants,such as vitamin A, vitamin E, and tocopherols/tocotrienols, andstatus of oxidation (peroxide and anisidine value) (Johansson andothers 1995b) as antioxidants can reduce the formation while theoxidized fat can increase it. Other researchers reported that fatsalso have an enhancing influence on the yield of HAAs in modelsystems after the addition of iron ions (Fe2+ or Fe3+), probably byfree radicals formed during thermally induced lipid oxidation. Inthis model system, the level of MeIQx formed was nearly dou-bled, probably due to iron-catalyzed lipid peroxidation and, thus,formation of free radicals (Felton and others 2000).

Most authors assume that the heat transfer is improved with anincrease in the fat content to an optimal fat–water ratio (Hwangand Ngadi 2002) leading to a shortening of the cooking time. Inthis study using high fat meat emulsions, the authors observed thatthe activation energy of HAA formation was reduced at high-fat



levels due to a more thermodynamically favored reaction (Hwangand Ngadi 2002). An increase in the fat content over the optimalratio results in a decreased HAA concentration (Persson and others2008; Abdulkarim and Smith 1998).

Moisture content and aw-valueWater serves as a reaction and transport medium during the

heating of meat and meat products. With water, the precursor sub-stances creatine/creatinine, amino acids, glucose, and so on, canbe transported to the surface of the product (Persson and others2002). At the surface, these substances are exposed to higher tem-peratures and so contribute to the formation of HAAs (Overvikand others 1989). The formation of mutagenic substances couldbe significantly reduced by reducing water vaporization during thecooking process (Persson and others 2004). The mutagenic activityis reduced with a reduction of the water content and an increasein the fat content; the capacity of the transport medium declinesand the precursors become diluted (Knize and others 1985).

An increase in water-holding capacity could, thus, be achievedby the addition of water-binding substances into minced meat(Persson and others 2003b, 2004; Wang and others 1982). Theauthors showed that with the addition of a tripolyphosphate plussodium chloride mixture (Persson and others 2003b), soy protein(Wang and others 1982) or carbohydrates (Shin and others 2003,2004; Persson and others 2004) to minced meat products, waterwas bound, leading to a reduction in HAA formation. Wrap-ping hamburgers with carrageenan had a similar effect (Schoch2003). The extreme dehydration obtained with the hot-air jetsresulted in a low water activity and slowed down the formationof IQx, MeIQx and, particularly, 4,8-DiMeIQx compared withsuperheated steam treatments (Kondjoyan and others 2010a,b).But the reverse effect was detected for PhIP concentrations whichincreased (Kondjoyan and others 2010b). In pan-fried bacon, theheat treatment of bacon with water activity around 0.93 caused ahigh content of MeIQx (Gibis and others 2015).

Antioxidants and reducing agentsNext to physical influences (temperature, time, and prepara-

tion method), added substances, such as nitrite, antioxidants, orspices, also showed an inhibiting effect on HAA formation assummarized for beef products as an example in (Table 6). Bothmain components such as lipids and proteins may be oxidized in aseries of radical reactions that include steps of initiation, propaga-tion, and termination with simultaneous formation of free radicals(Weiss and others 2010). The antioxidants did not only inhibitfat oxidation. It is known that free radicals may be involved inthe mechanism of HAA generation and Maillard reaction. Theantioxidants showed that they would scavenge the free radicalsand reduce HAA formation, and this could inhibit the radicalreactions in HAA formation, which was shown in an electronparamagnetic resonance experiment (Kikugawa 1999). Depend-ing on the substances added, HAA formation could be stimulatedor inhibited. When nitrite was added to meat products, severalHAAs, such as Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2, and AαC,were transformed under acidic conditions (pH 2) into their hy-droxy derivatives in model systems and so lost their mutagenicactivity (Furihata and Matsushima 1986). Nitrite is known as anadditive with properties as a reducing agent, at low pH values, thatcan reduce the concentration of non-IQ-type HAAs in modelsystems (Tsuda and others 1985; Shin 2005). Nitrite can also reactwith the amino acid cysteine causing the formation of an antiox-idant, and free radicals can, thus, be blocked (Shin 2005). The

generation of IQ-compounds is reduced by the addition of to-copherol (vitamin E) (Balogh and others 2000; Lan and others2004). Tocopherol is a naturally occurring antioxidant. The inhi-bition is due to the direct blocking of free radicals (Shin 2005). Inaddition, the breakdown products of tocopherol react with pre-cursors, which are then no longer available for the formation ofmutagens. Ascorbic acid or sodium ascorbate as a naturally occur-ring antioxidant and reductone, respectively, could also similarlybring about an HAA reduction (Kato and others 2000; Kiku-gawa and others 2000; Dundar and others 2012; Wong and others2012). A moderate inhibitory effect (approximately 20%) on theformation of PhIP, 4,8-DiMeIQx, and MeIQx was found forwater-soluble vitamins, such as niacin and ascorbic acid; whereaspyridoxamine reduced the concentrations of all 3 HAAs by ap-proximately 40% (Wong and others 2012). This study showedthat pyridoxamine reduced the level of PhIP significantly by trap-ping the phenylacetaldehyde and reacted with the latter to forma pyridoxamine-phenylacetaldehyde adduct which was confirmedby using LC–ESI–MS/MS and NMR spectroscopy (Wong andothers 2012). Rosemary extract reduced the content of HAAs infried beef patties (Puangsombat and Smith 2010; Damašius andothers 2011; Gibis and Weiss 2012). An inhibiting effect of thetomato carotenoid fraction on the formation of imidazoquino-lines (IQx, MeIQx, and DiMeIQx) was reported in model sys-tems containing freeze-dried bovine meat juice (Vitaglione andothers 2002). Using carotenoid extract at a concentration of 1000mg/kg, inhibitions of 13% of MeIQx and of 5% of 4,8-DiMeIQxin the meat juice model system were observed. The effect of themain tomato flavonoid, quercetin, gave an inhibition of MeIQxformation between 9% and 57% with a maximum effect of 67%at 10 mg/kg (Vitaglione and others 2002).

Butylated hydroxyanisole (BHA) is a synthetic antioxidant. Itwas seen in a study that the HAA level could be lowered by 40%with BHA (Weisburger 2005). A few years later, this inhibitoryeffect was confirmed and it was discovered that other phenolicantioxidants, such as epigallocatechin gallate and sesamol (Oguriand others 1998), had a positive effect on the reduction of HAAformation (Weisburger and others 1994).

Extracts of vegetables and fruit could also lead to a reduction inmutagenic activity (Britt and others 1998). Next to pure antioxi-dants, extracts with polyphenols present in plants were particularlyeffective (Ahn and Gruen 2005b; Cheng and others 2007; Gibisand Weiss 2012; Liao and others 2011a). The levels of differentHAAs, such as MeIQx or PhIP, can be lowered by heating modelsystems with polyphenols, such as quercetin, rutin, catechin, cat-echin gallate, and n-propyl gallate (Arimoto-Kobayashi and others2003; Ahn and Gruen 2005b). The results indicated that phe-nols having 2 hydroxy groups at meta positions of the aromaticring were the most efficient inhibitors. The presence of alkyl orcarboxylic groups as additional substituents in the aromatic ring re-duced the inhibitory effect slightly (Arimoto-Kobayashi and others2003). On the other hand, the introduction of additional hydroxyand amino groups mostly cancelled the inhibitory effect, whichwas also mostly absent in ortho and para dihydroxy derivatives;in complex phenols, the presence of several rings with oppositeeffects produced a reduced inhibitory effect (Arimoto-Kobayashiand others 2003). An isotope-labeling study showed that all frag-ments contained had phenylalanine as the origin. The reactionemployed phenylacetaldehyde and epigallocatechin gallate, whichfurther confirmed the ability

heterocyclicaromaticaminesincookedmeat products:causes...

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