Effect of the pyrrole polymerization mechanism on the antioxidative activity of nonenzymatic browning reactions

Article abstract of DOI:10.1021/jf034369u

The present investigation was undertaken to study how the antioxidative activity (AA) of nonenzymatic browning reactions is changing at the same time that the browning (by the pyrrole polymerization mechanism) is being produced. The antioxidative activiti

Full text of DOI:10.1021/jf034369u

J. Agric. Food Chem. 2003, 51, 5703−5708 5703
Effect of the Pyrrole Polymerization Mechanism on the
Antioxidative Activity of Nonenzymatic Browning Reactions
FRANCISCO J. HIDALGO, FAÄ TIMA NOGALES, AND ROSARIO ZAMORA*
Instituto de la Grasa, CSIC, Avenida Padre Garc´ıa Tejero 4, 41012 Seville, Spain
The present investigation was undertaken to study how the antioxidative activity (AA) of nonenzymatic
browning reactions is changing at the same time that the browning (by the pyrrole polymerization
mechanism) is being produced. The antioxidative activities of eight model pyrroles (pyrrole,
1-methylpyrrole, 2,5-dimethylpyrrole, 1,2,5-trimethylpyrrole, 2-acetylpyrrole, 2-acetyl-1-methylpyrrole,
pyrrole-2-carboxaldehyde, and 1-methyl-2-pyrrolecarboxaldehyde) as well as the browning reaction
of 2-(1-hydroxyethyl)-1-methylpyrrole (HMP) and the dimer (DIM) produced during HMP browning
were determined. The results obtained suggest that the AAs observed in nonenzymatic browning
reactions are the result of the AAs of the different oxidized lipid/amino acid reaction products formed.
Thus, the different pyrrole derivatives produced in these reactions had different AAs, and the highest
AAs were observed for alkyl-substituted pyrroles without free R-positions. Because some of these
pyrrole derivatives are implicated in nonenzymatic browning production and this browning production
implies the loss of hydroxyl groups and the transformation of some pyrroles with one type of substitution
into others, changes in AA during browning production were observed, and the resulting DIM derivative
was more antioxidant than HMP or higher polymers. These results explain the AA observed in fatty
acid/protein mixtures after slight oxidation and suggest that, when the pyrrole polymerization
mechanism is predominant, slightly browned samples may be more antioxidant than samples in which
nonenzymatic browning has been highly developed.
KEYWORDS: Carbonyl-amine reactions; lipid oxidation; Maillard reaction; natural antioxidants; non-
enzymatic browning; pyrrole derivatives; pyrrole polymerization
INTRODUCTION
ponents, normally present in foods, that possess antioxidative
properties. In this context, the antioxidative properties of
Maillard reaction products have long been known (11-13) and,
more recently, also the antioxidative activity of the nonenzy-
matic browned products formed in oxidized lipid/protein reac-
tions (14-16). Nevertheless, neither the relative antioxidative
activities of many of the different oxidized lipid/amino acids
reaction products (OLAARPs) nor how these activities are
changing in parallel to the oxidative process are known.
Lipids are important components that contribute very sig-
nificantly to the nutritional and sensory value of almost all kinds
of foods. This contribution is predominantly related to the
contents, distribution in the food matrix, chemical composition,
and reactivity of the lipids, as well as to their changes due to
processing, and the interactions with other food components
(1). When lipids are oxidized, sensory quality and nutritive value
of food products are deteriorated. For this reason, lipid oxidation
is a great economic concern to the food industry, and, therefore,
numerous studies have been carried out to control the implicated
processes (2-5).
Nowadays, antioxidants are commonly used as food additives
to extend the shelf life of oils and fatty foods during storage
and processing. The antioxidants to be used are determined by
various factors including legislation, effectiveness, and cost. In
addition, consumer preference for natural additives has encour-
aged the development of natural antioxidants. Thus, much
research has been conducted to find safe antioxidants with high
activity from natural resources (6-10). Furthermore, other
studies have been dedicated to isolate and characterize com-
Different studies have shown that when the lipid oxidation
process takes place in the presence of proteins, OLAARPs are
always produced (17). In fact, OLAARPs play a significant role
in the nonenzymatic browning process, and model studies
carried out in this laboratory have pointed to a pyrrole formation
and polymerization mechanism (Figure 1) as being responsible,
at least partially, for the nonenzymatic browning produced as a
consequence of oxidized lipid/amino acid reactions (18). The
key intermediates in this mechanism are N-substituted 2-(1-
hydroxyalkyl)pyrroles (I). These compounds have been shown
to be produced in the reaction of 4,5-epoxy-2-alkenals with the
amino groups of amino acids and proteins (19, 20), and their
formation is always accompanied by the production of N-
substituted pyrroles (II). These last compounds are relatively
stable and have been found in more than 20 fresh food products,
* Author to whom correspondence should be addressed [telephone
+(34) 954 611550; fax +(34) 954 616790; e-mail rzamora@ig.csic.es].
10.1021/jf034369u CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/12/2003

5704 J. Agric. Food Chem., Vol. 51, No. 19, 2003
Hidalgo et al.
Figure 1. Mechanism for nonenzymatic browning produced as a
Figure 2. Structures of the different pyrrole derivatives employed in this
study. Abbreviations: DIM, dimer produced in the polymerization reaction
of HMP; HMP, 2-(1-hydroxyethyl)-1-methylpyrrole; TRI, trimer produced
in the polymerization reaction of HMP; TET, tetramer produced in the
polymerization reaction of HMP.
consequence of 2-(1-hydroxyalkyl)pyrrole polymerization.
including meats, fishes, vegetables, and nuts (21). However,
the N-substituted 2-(1-hydroxyalkyl)pyrroles are unstable and
polymerize rapidly and spontaneously to produce brown mac-
romolecules with fluorescent melanoidin-like characteristics
(22).
According to this mechanism, hydroxylalkylpyrroles with at
least one unsubstituted R-position are produced in a first step,
and these monomers evolve alkyl-substituted pyrroles with no
free R-positions. The present study was undertaken to analyze
comparatively the antioxidative activities of different pyrrole
derivatives, including those formed in the first steps of pyrrole
polymerization, in an attempt to clarify how the antioxidative
activity of nonenzymatic browning reactions is changing at the
same time that the browning (by the pyrrole polymerization
mechanism) is being produced.
aqueous sodium hydroxide solution was added until a clear solution
was obtained, and the mixture was diluted with oxygen-free borate
buffer (50 mM, pH 9.0) to 25 mL and made up to 50 mL with oxygen-
free, distilled water. To determine the antioxidative activity, the sample
(60 µL of a solution containing 0.5-10 mg/mL in 60% ethanol) was
added to a solution of oxygen-saturated phosphate buffer (3 mL of 0.2
M, pH 6.75), hydrogen peroxide (100 µL of 16 mM), ferrous sulfate
(100 µL of 16 mM containing 15 mM EDTA), and linolenic acid
substrate (1.0 mL) and incubated for 10 min at room temperature. After
that time, 1 mL of the obtained solution was pipetted into disposable
cuvettes containing a solution (2.0 mL) of the color reagent consisting
of dimethylformamide (8%), Triton X-100 (1.4%), hemoglobin (56 mg/
L), and benzoyl leucomethylene blue (130 µM) in phosphate buffer
(0.2 M, pH 5.0). After an incubation time of 30 min, the absorbance
was measured at 666 nm against the buffer/color reagent blank. The
results were related to the absorption of an aqueous standard solution
of Trolox (1 mM) and were expressed as Trolox equivalent (TE) values.
Development of Nonenzymatic Browning by Pyrrole Polymer-
ization. Methanolic solutions of HMP and DIM (10 mg) were pipetted
into test tubes and taken to dryness to form a thin film. These tubes
were incubated under an inert atmosphere at 37 °C in the dark for
different periods of time. At the end of the incubation period, the sample
was dissolved in methanol (1 mL) and the color determined. Aliquots
of these solutions were also employed for GC-MS analyses and
antioxidative activity determinations.
EXPERIMENTAL PROCEDURES
Materials. Pyrrole, 1-methylpyrrole, 2,5-dimethylpyrrole, 1,2,5-
trimethylpyrrole, 2-acetylpyrrole, 2-acetyl-1-methylpyrrole, pyrrole-2-
carboxaldehyde, and 1-methyl-2-pyrrolecarboxaldehyde were purchased
from Aldrich (Milwaukee, WI). Structures for the different pyrroles
employed in this study are collected in Figure 2. Other reagents and
solvents were of analytical grade and were purchased from reliable
commercial sources.
2-(1-Hydroxyethyl)-1-methylpyrrole (HMP) and its dimer produced
during nonenzymatic browning, 1,1′-dimethyl-5-ethylidene-2,2′-dipyr-
rylmethylmethene (DIM), were prepared from 2-acetyl-1-methylpyrrole
according to a previously described procedure (22), which was modified.
Briefly, 2-acetyl-1-methylpyrrole (4 mmol) was reduced with sodium
borohydride (4 mmol) in methanol (5 mL) for 1.5 h at room
temperature. The reaction produced quantitatively HMP, but this
compound partially polymerized during column chromatography frac-
tionation on silica gel, and DIM was also isolated. Column chroma-
tography was carried out using diethyl ether/hexane (1:1) as eluent and
was followed by thin-layer chromatography with diethyl ether/hexane
(7:3). Identities of HMP (Rf 0.46) and DIM (Rf 0.38) were confirmed
Color Determination. The colors of the solutions were determined
spectrophotometrically using a Shimadzu UV-2401 PC UV-vis spec-
trophotometer. Color differences (∆E) at the different periods of time
were calculated from the determined CIELAB L* a* b* values
according to Hunter (25)
∆E ) [(L* - 100)2 + (a*)2 + (b*)2]1/2
by referring the determined values to an ideal colorless solution of L*
) 100 and a* ) b* ) 0.
1
by H and 13C nuclear magnetic resonance (NMR) spectroscopy and/
or gas chromatography-mass spectrometry (GC-MS).
GC-MS. Incubated samples (100 µL), containing cis-3-nonen-1-ol
(25 µL of a solution of 1.35 mg/mL in methanol) as internal standard,
were studied by GC-MS. GC-MS analyses were conducted with a
Hewlett-Packard 6890 GC Plus coupled with an Agilent 5973 MSD
(mass selective detector, quadrupole type). A fused-silica HP5-MS
capillary column (30 × 0.25 mm i.d.; coating thickness ) 0.25 µm)
Measurement of Antioxidative Activity in Pyrroles. Antioxidative
activity was determined following a described procedure (23, 24), which
was modified. Briefly, a linolenic acid substrate solution was prepared
by adding linolenic acid (0.125 mL) to a mixture of oxygen-free borate
buffer (2.5 mL of 50 mM, pH 9.0) and Tween 20 (0.125 mL). Then,

Pyrrole Polymerization and Antioxidative Activity
J. Agric. Food Chem., Vol. 51, No. 19, 2003 5705
Table 1. Antioxidative Activity of Pyrrolesa
concentration
compound
0.5 mg/mL (30 µg)
2 mg/mL (120 µg)
10 mg/mL (600 µg)
pyrrole
0.10 ± 0.02b
0.09 ± 0.02b
0.90 ± 0.13c
1.01 ± 0.02c
0.02 ± 0.05b
0.01 ± 0.06b
0.04 ± 0.11b
0.05 ± 0.03b
0.31 ± 0.04b
0.28 ± 0.04bc
0.87 ± 0.09d
0.91 ± 0.08d
0.18 ± 0.03bc
0.13 ± 0.01c
0.20 ± 0.03bc
0.24 ± 0.11bc
0.72 ± 0.02b
0.64 ± 0.07b
1.03 ± 0.11c
1.12 ± 0.06c
0.28 ± 0.05d
0.29 ± 0.03d
0.37 ± 0.05d
0.30 ± 0.09d
1-methylpyrrole
2,5-dimethylpyrroleb
1,2,5-trimethylpyrrolec
2-acetylpyrrole
2-acetyl-1-methylpyrrole
pyrrole-2-carboxaldehyde
1-methyl-2-pyrrolecarboxaldehyde
a Values are mean ± SE for three experiments and are given in Trolox equivalents (TE). The amount of compound introduced in the assay is given in parentheses.
Means in the same column with different letters are significantly different (p < 0.05). b Its antioxidative activities were 0.87 and 0.20 TE at 0.1 and 0.001 mg/mL, respectively.
c Its antioxidative activities were 0.93 and 0.31 TE at 0.1 and 0.001 mg/mL, respectively.
was used. Working conditions were as follows: carrier gas, helium (1
mL/min at constant flow); injector, 250 °C; oven temperature, from
70 (1 min) to 240 ° C at 5 °C/min and then to 325 °C at 10 °C/min;
transfer line to MSD, 280 °C; ionization EI, 70 eV.
Measurement of Antioxidative Activity during Nonenzymatic
Browning. Antioxidative activity was determined as described previ-
ously by using the incubated samples (100 µL), which were diluted
with 150 µL of methanol/water (3:2). Thirty microliter aliquots of the
diluted samples, which contained 120 µg of the tested compounds, were
employed for antioxidative activity determinations.
and 1-methylpyrrole at 0.5 mg/mL. However, although this
antioxidative activity increased with concentration, this increase
was lower than that exhibited by pyrrole and 1-methylpyrrole,
and the antioxidative activity exhibited by these oxygenated
compounds at 10 mg/mL was ∼0.3 TE. All of these results
suggested that the antioxidative activity of a nonenzymatic
browning reaction might be changing at the same time that the
different pyrroles are either being produced or disappearing to
evolve higher polymers.
Statistical Analysis. All results are expressed as mean values (
standard error (SE) of three independent experiments for model pyrroles
and, at least, six independent experiments for pyrrole polymerization.
Statistical comparisons among different groups were made using
ANOVA. When significant F values were obtained, group differences
were evaluated by the Student-Newman-Keuls test (26). All statistical
procedures were carried out using Primer of Biostatistics: The Program
(McGraw-Hill, Inc., New York). Significance level is p < 0.05 unless
otherwise indicated.
Change in the Antioxidative Activity during the Polym-
erization of HMP. HMP is an ideal model to study non-
enzymatic browning because the mechanism for browning
production has been well characterized (22) and it takes place
at a relatively slow rate. Figure 3A shows the GC chromatogram
of pure HMP. Although this compound was obtained with a
high purity, as indicated by both 1H and 13C NMR spectroscopy,
its GC-MS spectra presented several peaks. Thus, in addition
to the two peaks corresponding to HMP (one corresponding to
the compound dehydrated in the injector port of the chromato-
graph and the other corresponding to HMP itself), some small
peaks corresponding to dimers (DIM), trimers (TRI), and
tetramers (TET) could also be observed. They were likely
produced in the injector port of the chromatograph. Mass spectra
of HMP as well as DIM, TRI, and TET are collected in Figure
4.
RESULTS
Measurement of Antioxidative Activity in Pyrroles. Eight
different model pyrroles were assayed for antioxidative activity.
They included both unsubstituted and N-substituted pyrroles as
well as pyrroles with alkyl, aldehyde, and ketone groups at
position 2 or 5 of the pyrrole ring. Their antioxidative activities
in the assayed system are shown in Table 1. There was a very
different behavior between pyrroles with no free R-position and
those in which at least one of the R-positions was unsubstituted.
The highest antioxidative activity was exhibited by 2,5-
dimethylpyrrole and 1,2,5-trimethylpyrrole, both having no free
R-positions. Both compounds had similar antioxidative activities
among them, which were independent of concentration in the
range of 0.5-10 mg/mL, and all obtained values were similar
to that of 1 mM trolox (TE ) 1). Their antioxidant activities
decreased at lower concentrations and were 0.87 and 0.20 TE
for 2,5-dimethylpyrrole at 0.1 and 0.001 mg/mL, respectively,
and 0.93 and 0.31 TE for 1,2,5-trimethylpyrrole at 0.1 and 0.001
mg/mL, respectively. The next stronger antioxidants were
pyrrole and 1-methylpyrrole, both unsubstituted at the R-posi-
tion. These compounds exhibited an almost negligible antioxi-
dative activity when assayed at 0.5 mg/mL, but their antioxi-
dative activities increased with concentration, and ∼0.7 TE
values were obtained when assayed at 10 mg/mL. Finally, the
existence of an oxygenated group decreased the antioxidative
activity of pyrrole derivatives, and the lowest antioxidative
values were observed for 2-acetylpyrrole, 2-acetyl-1-methylpyr-
role, pyrrole-2-carboxaldehyde, and 1-methyl-2-pyrrolecarbox-
aldehyde. These last compounds exhibited similar antioxidative
activities among them, and they were also similar to pyrrole
When HMP was incubated under an inert atmosphere in the
dark at 37 °C, the chromatograms changed slightly and the
decrease of the monomers was accompanied by the formation
of polymers (Figure 3B). Figure 5 shows the evolution of HMP,
DIM, TRI, and TET as a function of incubation time. HMP
decreased almost linearly for the first 5 h and then more slowly.
After 24 h, the concentration of HMP was ∼50% of the initial
and it did not seem to experience any changes for the next 6 h.
This decrease in HMP was a consequence of its polymerization
and, therefore, polymer formation was observed. Thus, increases
for TRI and TET concentrations were observed during the whole
incubation period. However, DIM concentration increased
significantly for the first 2-3 h and then decreased to a value
that was slightly lower than the initial one. These results
suggested that HMP produced DIM in a first step and that both
HMP and DIM continued polymerizing to produce TRI, TET,
and higher polymers. In addition, the polymerization seemed
to be produced mainly on the DIM rather than in higher
polymers.
These results were confirmed when the changes suffered by
DIM upon incubation were studied. Thus, Figure 3C shows
the GC-MS chromatogram corresponding to untreated DIM. The
chromatogram showed the absence of monomers but the

5706 J. Agric. Food Chem., Vol. 51, No. 19, 2003
Hidalgo et al.
Figure 4. Mass spectra of (A) 2-(1-hydroxyethyl)-1-methylpyrrole (HMP),
(B) dimer produced in the polymerization reaction of HMP (DIM), (C) trimer
produced in the polymerization reaction of HMP (TRI), and (D) tetramer
produced in the polymerization reaction of HMP (TET). Structures for the
different compounds are given in Figure 2.
Figure 3. Total ion GC-MS chromatogram obtained for (A) pure 2-(1-
hydroxyethyl)-1-methylpyrrole (HMP), (B) HMP incubated for 29 h, (C)
unincubated dimer produced in the polymerization reaction of HMP (DIM),
and (D) DIM incubated for 29 h. Abbreviations: DIM, dimer produced in
the polymerization reaction of HMP; HMP, 2-(1-hydroxyethyl)-1-meth-
ylpyrrole; IS, internal standard (cis-3-nonen-1-ol); TRI, trimer produced in
the polymerization reaction of HMP; TET, tetramer produced in the
polymerization reaction of HMP. Structures for the different compounds
are given in Figure 2.
Figure 5. Time course of monomer disappearance and polymer formation
during 2-(1-hydroxyethyl)-1-methylpyrrole (HMP) incubation under an inert
atmosphere in the dark at 37 °C. Compounds determined were (0) 2-(1-
hydroxyethyl)-1-methylpyrrole (HMP), (O) dimer produced in the polym-
erization reaction of HMP, (4) trimer produced in the polymerization
reaction of HMP, and (3) tetramer produced in the polymerization reaction
of HMP. Values are the mean ± SE for six experiments. Structures for
the different compounds are given in Figure 2.
presence of a certain quantity of TRI. These last polymers might
be present in the initial DIM because these compounds and
higher polymers are not easily fractionated by absorption
chromatography. In addition, it is unclear that DIM may be
converted into TRI (see below). When DIM was incubated under
an inert atmosphere in the dark at 37 °C, it rapidly polymerized,
analogously to HMP (Figure 3D). Figure 6 shows the evolution
of HMP, DIM, TRI, and TET as a function of incubation time.
Analogously to HMP, DIM also decreased linearly for the first
5 h and, then, much more slowly. This decrease was not
accompanied by an increase in TRI, which also decreased with
time. On the other hand, an increase in TET was observed. TET
increased almost linearly for the first 5 h; its concentration then
stabilized for a long time period and, finally, decreased. These
results suggested a transformation of DIM into TET and, then,
into higher polymers.
color development in both HMP and DIM incubations. Color
difference increased linearly (r ) 0.986, p < 0.0001) in HMP
incubated under an inert atmosphere in the dark at 37 °C. This
increase was not so clearly observed in DIM, although it
increased in a way parallel to DIM disappearance (Figure 6).
In fact, DIM decrease and color formation in DIM incubation
were correlated (r ) -0.95, p < 0.001).
These conversions of some pyrroles into others were very
related to the changes produced in the antioxidative activities
exhibited by HMP and DIM when incubated under an inert
atmosphere in the dark at 37 °C (Figure 8). Thus, HMP and
DIM exhibited antioxidative activities of 0.30 and 0.69 TE,
respectively, when employing 120 µg of sample. The antioxi-
dative activity of HMP increased almost linearly for the first 5
h (TE values were doubled after that time) and, then, decreased
to arrive at 1.5 times the initial TE value. This behavior seemed
The polymerizations produced in all of these reactions,
confirmed by GC-MS for small polymers and suggested for
bigger ones, were also in agreement with color formation in
these reactions, which has been shown to be a consequence of
the formation of long-chain polymers (22). Figure 7 shows the

Pyrrole Polymerization and Antioxidative Activity
J. Agric. Food Chem., Vol. 51, No. 19, 2003 5707
with two pyrrole rings without oxygenated functions and one
of them with no free R-position, DIM was ∼2.5 times more
antioxidative than HMP. The incubation of DIM under an inert
atmosphere in the dark at 37 °C produced a decrease in the
antioxidative activity, which was parallel to DIM disappearance
observed by GC-MS (Figure 6). In fact, both decreases were
correlated (r ) 0.937, p ) 0.0018). These results confirmed
that DIM was a better antioxidant than higher polymers.
DISCUSSION
The results obtained in this study suggest that the mechanisms
implicated in nonenzymatic browning are closely related to the
antioxidative activity exhibited by OLAARPs. Among the
different products formed in the reactions between oxidized
lipids and amino acids and proteins, different pyrrole derivatives
have been shown to be produced in the reaction of amino groups
with 4,5-epoxy-2-alkenals (27), 4-hydroxy-2-alkenals (28),
4-oxo-2-alkenals (29), unsaturated epoxyoxo fatty acids (30),
and lipid hydroperoxides (31), among others. All of these types
of pyrrole derivatives with different substituents in the pyrrole
ring also play a significant role in the antioxidative activity of
foods. However, the different pyrrole derivatives have different
antioxidative activities, and the highest antioxidative activities
have been observed for nonoxygenated pyrrole derivatives with
no free R-positions.
Figure 6. Time course of reactions produced during incubation under an
inert atmosphere in the dark at 37 °C of the dimer produced in the
polymerization reaction of 2-(1-hydroxyethyl)-1-methylpyrrole. Compounds
determined were (0) 2-(1-hydroxyethyl)-1-methylpyrrole (HMP), (O) dimer
produced in the polymerization reaction of HMP, (4) trimer produced in
the polymerization reaction of HMP, and (3) tetramer produced in the
polymerization reaction of HMP. Values are the mean ± SE for six
experiments. Structures for the different compounds are given in Figure
2.
Because some of these pyrrole derivatives are implicated in
nonenzymatic browning production and this browning produc-
tion implies the loss of hydroxyl groups and the transformation
of some pyrroles with one type of substitution into others,
changes in antioxidative activity during browning production
should be expected. The results obtained in this study agree
with this hypothesis. In addition, the above results suggest that
not all of the different polymers produced have similar anti-
oxidative activities, although all of them are believed to be
similarly substituted at the R-positions. In fact, the dimer was
found to be the most antioxidative derivative. The reason for
the observed differences in the antioxidative activities among
the different polymeric pyrroles is unclear at present.
Figure 7. Color development in the incubation under an inert atmosphere
in the dark at 37 °Cof (0) 2-(1-hydroxyethyl)-1-methylpyrrole (HMP) and
(O) the dimer produced in the polymerization reaction of HMP. Values
are the mean ± SE for six experiments.
The above results explain the antioxidative activity observed
in polyunsaturated fatty acid/protein mixtures after slight
oxidation (32). They also suggest that slightly browned samples
may be more antioxidative than samples in which nonenzymatic
browning has been highly developed, an effect that has been
observed, for example, in coffee roasting (33) or black tea
beverage storage (34). In addition, all of these results point out
that the antioxidative activity observed in a nonenzymatic
browning reaction is the sum of the antioxidative activities of
the different compounds present in the sample.
Figure 8. Antioxidative activity of the incubations under an inert
atmosphere in the dark at 37 °Cof (0) 2-(1-hydroxyethyl)-1-methylpyrrole
(HMP) and (O) the dimer produced in the polymerization reaction of HMP.
Values are the mean ± SE for six experiments.
ABBREVIATIONS USED
DIM, dimer produced in the polymerization reaction of HMP;
EDTA, ethylenediaminetetraacetic acid; GC-MS, gas chroma-
tography-mass spectrometry; HMP, 2-(1-hydroxyethyl)-1-
methylpyrrole; NMR, nuclear magnetic resonance; OLAARPs,
oxidized lipid/amino acid reaction products; TE, Trolox equiva-
lents; TET, tetramer produced in the polymerization reaction
of HMP; TRI, trimer produced in the polymerization reaction
of HMP.
to be a consequence of the different compounds that were being
produced in the reaction. Thus, during the first few hours of
incubation, the formation of short polymers was favored (Figure
5). Because these compounds have no hydroxyl groups and
fewer free R-positions than monomers, they should be more
antioxidative. As the incubation progressed, these small poly-
mers were transformed into higher polymers. This increase in
polymer chain length or further reactions that may take place
in parallel to pyrrole polymerization should decrease the
antioxidative activity. These results are also in agreement with
those obtained during DIM incubation. As expected for a dimer
ACKNOWLEDGMENT
We are indebted to Jose´ L. Navarro for technical assistance.

5708 J. Agric. Food Chem., Vol. 51, No. 19, 2003
Hidalgo et al.
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Received for review April 11, 2003. Revised manuscript received July
2, 2003. Accepted July 6, 2003. This study was supported in part by
the Ministerio de Ciencia y Tecnolog´ıa of Spain (Project AGL2000-1615).
JF034369U