Contribution of lipid oxidation products to acrylamide formation in model systems

Article abstract of DOI:10.1021/jf073047d

The reactions of asparagine with methyl linoleate (1), methyl 13-hydroperoxyoctadeca-9,11-dienoate (2), methyl 13-hydroxyoctadeca-9,11- dienoate (3), methyl 13-oxooctadeca-9,11-dienoate (4), methyl 9,10-epoxy-13-hydroxy-11-octadecenoate (5), methyl 9,10-epoxy-13-oxo-11- octadecenoate (6), 2,4-decadienal (7), 2-octenal (8), 4,5-epoxy-2-decenal (9), and benzaldehyde (10) were studied to determine the potential contribution of lipid derivatives to acrylamide formation in heated foodstuffs. Reaction mixtures were heated in sealed tubes for 10 min at 180°C under nitrogen. The reactivity of the assayed compounds was 7 ? 9 > 4 > 2 ? 8 ～ 6 ? 10 ～ 5. The presence of compounds 1 and 3 did not result in the formation of acrylamide. These results suggested that α,β,γ, δ-diunsaturated carbonyl compounds were the most reactive compounds for this reaction followed by lipid hydroperoxides, more likely as a consequence of the thermal decomposition of these last compounds to produce α,β,γ,δ-diunsaturated carbonyl compounds. However, in the presence of glucose this reactivity changed, and compound 1/glucose mixtures showed a positive synergism (synergism factor = 1.6), which was observed neither in methyl stearate/glucose mixtures nor in the presence of antioxidants. This synergism is proposed to be a consequence of the formation of free radicals during the asparagine/glucose Maillard reaction, which oxidized the lipid and facilitated its reaction with the amino acid. These results suggest that both unoxidized and oxidized lipids are able to contribute to the conversion of asparagine into acrylamide, but unoxidized lipids need to be oxidized as a preliminary step.

Full text of DOI:10.1021/jf073047d

J. Agric. Food Chem. 2008, 56, 6075–6080 6075
Contribution of Lipid Oxidation Products to
Acrylamide Formation in Model Systems
ROSARIO ZAMORA AND FRANCISCO J. HIDALGO*
Instituto de la Grasa, Consejo Superior de Investigaciones Cient´ıficas, Avenida Padre Garc´ıa Tejero 4,
41012 Seville, Spain
The reactions of asparagine with methyl linoleate (1), methyl 13-hydroperoxyoctadeca-9,11-dienoate
(2), methyl 13-hydroxyoctadeca-9,11-dienoate (3), methyl 13-oxooctadeca-9,11-dienoate (4), methyl
9,10-epoxy-13-hydroxy-11-octadecenoate (5), methyl 9,10-epoxy-13-oxo-11-octadecenoate (6), 2,4-
decadienal (7), 2-octenal (8), 4,5-epoxy-2-decenal (9), and benzaldehyde (10) were studied to
determine the potential contribution of lipid derivatives to acrylamide formation in heated foodstuffs.
Reaction mixtures were heated in sealed tubes for 10 min at 180 °C under nitrogen. The reactivity
of the assayed compounds was 7 . 9 > 4 > 2 . 8 ∼ 6 . 10 ∼ 5. The presence of compounds 1
and 3 did not result in the formation of acrylamide. These results suggested that R,ꢀ,γ,δ-diunsaturated
carbonyl compounds were the most reactive compounds for this reaction followed by lipid
hydroperoxides, more likely as a consequence of the thermal decomposition of these last compounds
to produce R,ꢀ,γ,δ-diunsaturated carbonyl compounds. However, in the presence of glucose this
reactivity changed, and compound 1/glucose mixtures showed a positive synergism (synergism factor
) 1.6), which was observed neither in methyl stearate/glucose mixtures nor in the presence of
antioxidants. This synergism is proposed to be a consequence of the formation of free radicals during
the asparagine/glucose Maillard reaction, which oxidized the lipid and facilitated its reaction with the
amino acid. These results suggest that both unoxidized and oxidized lipids are able to contribute to
the conversion of asparagine into acrylamide, but unoxidized lipids need to be oxidized as a preliminary
step.
KEYWORDS: Acrylamide; asparagine; carbonyl-amine reactions; decadienal; epoxydecenal; hydroper-
oxide; lipid oxidation; lipid oxidation products; Maillard reaction; 2-octenal
INTRODUCTION
pathways (18). Thus, among amino acid degradation studies,
different investigations have shown that suitable amino acids
may be converted into their corresponding Strecker aldehydes
by different tertiary lipid oxidation products at low or
moderate temperatures (19–23). In addition, certain secondary
lipid oxidation products may convert amino acids either into
their corresponding Strecker aldehydes or into vinylogous
derivatives depending on the amount of oxygen present in
the reaction (24, 25), therefore suggesting a potential route
for the contribution of these secondary lipid oxidation
products to acrylamide formation in thermally treated foods.
Acrylamide has been classified by the International Agency
for Research on Cancer as probably carcinogenic to humans
(1). In addition, neurological and reproductive effects of this
compound have also been described (1). Therefore, the detection
of this compound in a broad range of heated foods at concentra-
tions sometimes exceeding 1 ppm (2) has led to considerable
worldwide activity to investigate its origin and to reduce its
content in foods (3–16).
Nowadays, acrylamide is broadly accepted to be produced
at elevated temperatures and medium to low moisture
contents as a consequence of the asparagine degradation
produced by the Maillard reaction initiated by carbohydrates,
although a few studies have proposed the contribution of
lipids via the formation of acrolein (17). In addition, other
studies have shown that oxidized lipids compete very
efficiently with carbohydrates for carbonyl-amine reactions,
and the same products are frequently produced from either
carbohydrates or lipids by identical or very similar reaction
As a continuation of these last studies, the present investiga-
tion was undertaken to determine the different lipid oxidation
products that can contribute to the formation of acrylamide from
asparagine. Model reactions were carried out with both methyl
linoleate (1) and methyl stearate as well as with primary,
secondary, and tertiary products of the oxidation of compound
1. In addition, different mixtures of lipids and carbohydrates
were also studied to understand the reactions that take place in
real foodstuffs in which amino acids, lipids, and carbohydrates
are in close contact.
* Author to whom correspondence should be addressed [fax +(34)
954 616 790; e-mail fhidalgo@ig.csic.es].
10.1021/jf073047d CCC: $40.75  2008 American Chemical Society
Published on Web 07/15/2008

6076 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Zamora and Hidalgo
Figure 1. Chemical structures of the fatty acid derivatives employed in this study. They were derived from the ω6 linoleic acid. 1, Methyl linoleate; 2,
methyl 13-hydroperoxyoctadeca-9,11-dienoate; 3, methyl 13-hydroxyoctadeca-9,11-dienoate; 4, methyl 13-oxooctadeca-9,11-dienoate; 5, methyl 9,10-
epoxy-13-hydroxy-11-octadecenoate; 6, methyl 9,10-epoxy-13-oxo-11-octadecenoate; 7, 2,4-decadienal; 8, 2-octenal; 9, 4,5-epoxy-2-decenal. Also included
is the chemical structure of benzaldehyde (10), which was employed for comparison purposes.
MATERIALS AND METHODS
Methyl 13-hydroxyoctadeca-9,11-dienoate (3) was prepared by
reducing the 13-hydroperoxide of linoleic acid with sodium borohydride
and later esterification with diazomethane (26). Compound 3 was
purified by column chromatography on silica gel using hexane/diethyl
ether (7:3) as the eluent. This compound was obtained chromatographi-
cally pure. Additional confirmations of identity and purity were obtained
by 1D and 2D NMR. 13C NMR (CDCl3, 75.4 MHz) of compound 3:
δ 174.36 (C1), 135.84 (C12), 132.80 (C9), 127.71 (C10), 125.69 (C11),
72.87 (C13), 51.47 (OCH3), 37.21 (C14), 34.00 (C2), 31.72 (C16), 29.40
(C6), 28.99, 28.99, 28.89 (C4-C6), 27.61 (C8), 25.08 (C15), 24.82
(C3), 22.56 (C17), and 14.03 (C18). This spectrum was identical, for
example, to that previously collected by Ha¨ma¨la¨inen and Kamal-Eldin
(28).
Materials. All chemicals were purchased from Aldrich (Milwakee,
WI), Sigma (St. Louis, MO), Fluka (Buchs, Switzerland), or Merck
(Darmstadt, Germany) and were of analytical grade. Labeled [1,2,3-
13C3]acrylamide was obtained from Cambridge Isotope Laboratories,
Inc. (Andover, MA).
Lipids and Lipid Oxidation Products. Unoxidized lipids as well
as primary, secondary, and tertiary lipid oxidation products derived
from ω6 linoleic acid were employed in this study. The chemical
structures of the lipid derivatives employed in this study are given in
Figure 1. This figure also includes the chemical structure of benzal-
dehyde, which was employed for comparison purposes. On the contrary,
the chemical structure of methyl stearate has not been included. It is
identical to that of compound 1, but the fatty chain is saturated.
Methyl 13-oxooctadeca-9,11-dienoate (4) was prepared by oxidation
of 13-hydroxyoctadeca-9,11-dienoic acid with chromium trioxide and
later esterification with diazomethane (26). Compound 4 was purified
by column chromatography on silica gel using hexane/diethyl ether
(4:1) as the eluent. This compound was obtained chromatographically
pure and exhibited the previously described 1H and 13C NMR spectra
(26).
Methyl linoleate (1) (>98.5%) and methyl stearate (>99%) were
obtained from Fluka and were used without further purification.
Methyl 13-hydroperoxyoctadeca-9,11-dienoate (2) was prepared by
oxidation of the corresponding fatty acid with lipoxygenase following
a previously described procedure (26). The obtained hydroperoxide was
esterified with diazomethane and purified by column chromatography
on silica gel using hexane/diethyl ether (7:3) as the eluent. Compound
2 was obtained chromatographically pure. Additional confirmations of
identity and purity were obtained by 1D and 2D NMR. 13C NMR
(CDCl3, 75.4 MHz) of compound 2: δ 174.49 (C1), 133.96 (C9), 131.23
(C12), 130.11 (C11), 127.51 (C10), 86.85 (C13), 51.54 (OCH3), 34.06
(C2), 32.49 (C14), 31.72 (C16), 29.35, 29.03, 29.00, 28.87 (C4-C7),
27.69 (C8), 24.98 (C15), 24.85 (C3), 22.51 (C17), and 14.04 (C18).
This spectrum was identical to that previously described by Dussault
et al. (27).
Methyl 9,10-epoxy-13-oxo-11-octadecenoate (6) was prepared by
epoxidation of 4 with 3-chloroperoxybenzoic acid (29). Compound 6
was purified by column chromatography on silica gel using hexane/
diethyl ether (4:1) as the eluent. This compound was obtained
chromatographically pure and exhibited the previously described 1H
and 13C NMR spectra (26).
Methyl 9,10-epoxy-13-hydroxy-11-octadecenoate (5) was prepared
by reduction of 6 with sodium borohydride (26). Compound 5 was
purified by column chromatography on silica gel using hexane/diethyl

Acrylamide Symposium
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6077
ether (3:2) as the eluent. This compound was obtained chromatographi-
cally pure and exhibited the previously described 1H and 13C NMR
spectra (26).
2,4-Decadienal (7) (93%) was obtained from Aldrich. It was further
purified by column chromatography on silica gel 60 using hexane/
acetone (9:0.25) as solvent. The aldehyde recovered from the column
was chromatographically pure as determined by GC.
4,5-Epoxy-2-decenal (9) was prepared by epoxidation of 7 with
3-chloroperoxybenzoic acid (23). Compound 9 was purified by column
chromatography on silica gel using hexane/diethyl ether (95:5) as the
eluent. This compound was obtained chromatographically pure. Ad-
1
ditional confirmations of identity and purity were obtained by H and
13C NMR and GC-MS.
2-Octenal (8) and benzaldehyde (10) were used without further
purification.
All of these lipid derivatives, among other compounds, are produced
during oxidation of compound 1. Thus, the oxidation of 1 produces 2,
which is later decomposed to form 3, 4, 7, and 8, among other
compounds. Later oxidation of 3, 4, and 7 is the origin of 5, 6, and 9,
respectively. These pathways have been broadly studied (30, 31).
Additional pathways for the formation of some of these compounds
have also been described (30, 31), for example, the formation of 9 by
the degradation of an epoxyhydroperoxy fatty acid. These additional
pathways are also in agreement with the conclusions obtained in the
present study.
Asparagine/Lipid Reaction Mixtures. Model reactions were carried
out analogously to that used by Granvogl and Schieberle (8), with the
modifications described by Hidalgo and Zamora (25). Briefly, mixtures
of asparagine and the lipid derivative (75 µmol each) were singly
homogenized with 0.063-0.200 mm silica gel 60 (600 mg) (Macherey-
Nagel, Du¨ren, Germany), 60 µL of 0.3 M sodium phosphate, pH 6,
and 360 µL (31%) of water. Samples were heated under nitrogen at
180 °C in closed test tubes for 10 min. After cooling (15 min at
-20 °C), 60 µL of internal standard solution (0.1 mg/mL of labeled
[1,2,3-13C3]acrylamide in water) and 2 mL of 0.3 M sodium citrate
buffer, pH 2.2, were added. Suspensions were stirred for 1 min, the
supernatant was then filtered, and its acrylamide content was
determined.
Figure 2. Acrylamide produced in asparagine/lipid (1:1) model systems
heated at 180 °C for 10 min under nitrogen. Chemical structures for the
assayed lipids are given in Figure 1.
Quantification of acrylamide was carried out by preparing standard
curves of this compound in the 600 mg of silica gel and following the
whole procedure described above. For each curve, 15 different
concentration levels of acrylamide (0-200 µg) were used. Acrylamide
content was directly proportional to the acrylamide/internal standard
area ratio (r ) 0.999, p < 0.0001). The coefficients of variation at the
different concentrations were <10%. All data given are mean values
of, at least, two independent experiments.
RESULTS
Acrylamide Formation in Asparagine/Lipid Reaction
Mixtures. When heated with asparagine, different lipid oxida-
tion products converted this amino acid into acrylamide to some
extent (Figure 2). Figure 2 also includes the data obtained with
10, which is not a lipid oxidation product, for comparison
purposes.
The assayed unoxidized lipid (1) did not produce acrylamide
analogously to the asparagine heated in the absence of lipid
(control). Analogous results were also obtained with the
secondary and tertiary lipid oxidation products, which did not
have a carbonyl group. Thus, the hydroxydiene 3 was not able
to convert asparagine into acrylamide, and its epoxy derivative
5 produced <0.02% of acrylamide. On the other hand, the
hydroperoxide 2 and all of the lipid derivatives assayed having
a carbonyl group in their structures were able to convert
asparagine into acrylamine. The yield of acrylamide formation
depended on the lipid oxidation product employed and ranged
from the 4.7% obtained for compound 7 to the 0.08% obtained
for compound 6.
Among the long-chain and short-chain derivatives studied,
the highest reaction yields were obtained for the correspond-
ing R,ꢀ,γ,δ-diunsaturated carbonyl compounds (4 and 7,
respectively). In addition, the epoxidation of one of the
double bonds in both 4 and 7 to produce 6 and 9, respectively,
decreased considerably the reaction yield (between 5 and 8
times).
The yield of the hydroperoxide 2 was relatively high for
a long-chain lipid oxidation product (0.4%) and was only
lower than that obtained for the long-chain derivative 4
(0.6%).
Asparagine/Glucose/Lipid Reaction Mixtures. Model reactions
were carried out, and acrylamide was determined, analogously to the
above-described method for asparagine/lipid reaction mixtures, but 75
µmol of asparagine, 37.5 µmol of the glucose, and 37.5 µmol of the
lipid were employed. In addition, some mixtures also included 9.4 µmol
of BHT.
Analysis of Acrylamide in Model Systems. Acrylamide was
analyzed as the stable 2-bromopropenamide by gas chromatography-
mass spectrometry (GC-MS) using the method of Castle et al. (32)
with the modifications of Andrawes et al. (33). Briefly, 1 mL of the
supernatant was treated with 0.3 g of potassium bromide and 400 µL
of saturated bromine solution in water. After 1 h in the dark at 0 °C,
the excess of bromine was removed by the addition of 1 M sodium
thiosulfate until the solution became colorless, and the solution was
extracted with 1 mL of ethyl acetate/hexane (4:1). The organic layer
was finally dried with sodium sulfate, evaporated to a volume of ∼50
µL, treated with 50 µL of triethylamine, and analyzed by GC-MS.
The ions monitored for the identification of the analyte, 2-bromopro-
penamide, were [C3H4NO]+ ) 70, [C3H479BrNO]+ ) 149, and
[C3H481BrNO]+ ) 151, using m/z 149 for quantitation. The ions
monitored for identification of the internal standard (2-bromo-
[
13C3]propenamide) were [13C2H381Br]+ ) 110 and [13C3H481BrNO]+
) 154, using m/z 154 for quantitation. Mass spectra of both compounds
are collected, for example, by Pittet et al. (34). The separation of
acrylamide analyte after derivatization was performed on GC capillary
columns of middle to high polarity. GC-MS analyses were conducted
with a Hewlett-Packard 6890 GC Plus coupled with an Agilent 5973
MSD (mass selective detector-quadrupole type). In most experiments,
a 30 m × 0.25 mm i.d. × 0.25 µm HP5-MS capillary column was
used. Working conditions were as follows: carrier gas, helium (1 mL/
min at constant flow); injector temperature, 250 °C; oven temperature,
raised from 70 °C (1 min) to 240 at 5 °C/min and then to 325 °C at
10 °C/min; transfer line to MSD, 280 °C; and ionization EI, 70 eV.
Although short-chain aldehydes were more reactive than the
analogous long-chain ketone derivatives, the reaction yield for
short-chain aldehydes was low if the structure was not appropri-
ate. Thus, the yield of compound 8 was only 0.1%, despite the
difference between compounds 8 and 7 being only one double
bond. Furthermore, the reaction yield for 10, an aromatic
aldehyde, was 0.02%.

6078 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Zamora and Hidalgo
DISCUSSION
The results obtained in this study indicate that, in addition to
carbohydrates, different lipid oxidation products are able to
convert asparagine into acrylamide. This conversion is favored
by oxidized lipids having an R,ꢀ,γ,δ-diunsaturated carbonyl
group. The obtained results suggest that the diunsaturated
aldehyde 7 is more reactive than the diunsaturated ketone 4.
This is likely a consequence of both a higher reactivity of the
aldehyde group in comparison to the ketone group for this
reaction and the different solubilities of short-chain and long-
chain lipid oxidation products. Short-chain aldehydes are more
hydrophilic and, therefore, they should be much more easily in
close contact with asparagine. However, the structure of the
compound is much more important than the solubility, and short-
chain aldehydes not having the R,ꢀ,γ,δ-diunsaturated carbonyl
group produced very reduced amounts of acrylamide. Thus, for
example, an aromatic aldehyde, such as benzaldehyde, produced
only very small amounts of acrylamide.
When the R,ꢀ,γ,δ-diunsaturated carbonyl group suffered a
further oxidation step (such as in the formation of compounds
9 and 6), its reactivity for this reaction was reduced considerably
in comparison to their corresponding secondary lipid oxidation
products (compounds 7 and 4, respectively). However, the
reactivity of 9 for this reaction was still very high. Additional
studies are needed to investigate the reactivity of this aldehyde,
which might be related to a reversion of the epoxidation reaction
at high temperature. These studies are being developed at present
in this laboratory.
In addition to R,ꢀ,γ,δ-diunsaturated carbonyl compounds, the
diunsaturated hydroperoxide 2 was also very reactive for this
reaction. This may be a consequence of the easy decomposition
of hydroperoxides to produce R,ꢀ,γ,δ-diunsaturated carbonyl
compounds, among other compounds (30, 31). This result is
very important because oxidized lipids are usually present in
foods at lower concentrations than employed in this study.
However, the obtained results suggest that the presence of
R,ꢀ,γ,δ-diunsaturated carbonyl compounds is not necessary to
convert asparagine into acrylamide. The presence of the primary
lipid oxidation products, or even unoxidized lipids under
conditions that can be oxidized, is the only requisite needed to
observe a positive contribution of the lipids to acrylamide
formation during food heating.
These conclusions were confirmed when mixtures of lipids
and glucose were heated in the presence of asparagine. The
diunsaturated lipid 1 alone was found to be unable to convert
asparagine into acrylamide. However, the Maillard reaction
between asparagine and glucose is likely to produce free
radicals (35–37) that should oxidize compound 1 to 2. The
formation of this hydroperoxide 2 should be responsible for
the positive synergism observed in compound 1/glucose/
asparagine mixtures. On the contrary, methyl stearate, which
cannot be oxidized, did not exhibit any positive synergism.
Furthermore, the addition of an antioxidant to compound
1/glucose/asparagine mixtures inhibited the oxidation of the
unsaturated fatty ester and, therefore, mostly reduced the
synergism in those samples. Moreover, the production of free
radicals in compound 7/glucose/asparagine mixtures should
convert the R,ꢀ,γ,δ-diunsaturated aldehyde 7 into a much
less reactive derivative for this reaction, which is in agree-
ment with both the negative synergism observed in these
mixtures and the reduction of this negative synergism in the
presence of antioxidants.
Figure 3. Acrylamide produced in asparagine/glucose/lipid (2:1:1) and
asparagine/glucose/lipid/BHT (2:1:1:0.25) model systems heated at
180 °C for 10 min under nitrogen (mixture and mixt/BHT, respectively).
Acrylamide produced in binary mixtures (2:1) of asparagine with either
the lipid or glucose is also given for comparison. The lipids added were
A, methyl linoleate (1); B, methyl stearate; and C, 2,4-decadienal (7).
Acrylamide Formation in Asparagine/Glucose/Lipid Re-
action Mixtures. In foods, lipids are not the unique com-
pounds in contact with asparagine. Therefore, lipid/carbo-
hydrate interactions (see, for example, ref 18 for a recent
review) can play a role in the amount of acrylamide produced.
Figure 3A shows the acrylamide produced when asparagine
was heated in the presence of the unsaturated fatty ester 1,
glucose, the mixture of both compounds, and the mixture of
compound 1, glucose, and BHT. As described above, compound
1 did not produce acrylamide. On the other hand, the reaction
yield for glucose was 0.4%. However, when asparagine was
incubated in the presence of both compound 1 and glucose, a
positive synergism was observed, and acrylamide was produced
with a reaction yield of 0.6%. The synergism factor for this
mixture, which is defined as the ratio between the experimental
value and the value calculated considering the effect offered
by both components when tested individually, was 1.6. This
synergism was much reduced when the compound 1/glucose/
asparagine mixture was incubated in the presence of BHT
(synergism factor ) 1.1).
To obtain further confirmation that the oxidation of the
lipid was the responsible for the acrylamide produced, the
same mixtures were analyzed but methyl stearate was
employed in place of compound 1 (Figure 3B). Neither the
methyl stearate/glucose/asparagine mixture (synergism factor
) 1.0) nor the methyl stearate/glucose/asparagine/BHT
mixture (synergism factor ) 0.9) exhibited any increase in
the acrylamide determined.
Different from the behavior exhibited by compound 1 or by
methyl stearate, when compound 7 was heated with asparagine
in the presence of glucose, the amount as acrylamide decreased
and a negative synergism was observed for these two com-
pounds (Figure 3C). Thus, the acrylamide yield obtained for 7
alone (3.8%) decreased to 1.1% in the presence of glucose
(synergism factor ) 0.26). This decrease was lower in the
presence of BHT (synergism factor ) 0.36).
The effect of antioxidants on acrylamide formation in heated
foodstuffs is not completely clarified yet, and it may be related

Acrylamide Symposium
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6079
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All of these results suggest that both unoxidized and
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ACKNOWLEDGMENT
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Received for review October 16, 2007. Revised manuscript received
January 10, 2008. Accepted May 18, 2008. This study was supported
in part by the European Union (FEDER funds), the Junta de Andaluc´ıa
(Project P07-AGR-2846), and the Plan Nacional de I + D of the
Ministerio de Educacio´n y Ciencia of Spain (Project AGL2006-01092).
JF073047D