Bound malondialdehyde in foods: Bioavailability of the N-2-propenals of lysine

Article abstract of DOI:10.1021/jf025681r

The lipid peroxidation product malondialdehyde is mostly bound to proteins in foods as an N-2-propenal derivative that is released as N-ε-(2-propenal)lysine by digestive enzymes. N-2-Propenals have been identified as the major forms of malondialdehyde in

Full text of DOI:10.1021/jf025681r

6194 J. Agric. Food Chem. 2002, 50, 6194−6198
Bound Malondialdehyde in Foods: Bioavailability of the
N-2-Propenals of Lysine
JULIO GIROÄ N-CALLE,* MANUEL ALAIZ, FRANCISCO MILLAÄ N,
†
VALENTINA RUIZ-GUTIEÄ RREZ, AND EDUARDO VIOQUE
Instituto de la Grasa (Consejo Superior de Investigaciones Cient´ıficas),
Avenida Padre Garc´ıa Tejero 4, 41012 Sevilla, Spain
The lipid peroxidation product malondialdehyde is mostly bound to proteins in foods as an N-2-
propenal derivative that is released as N-ꢀ-(2-propenal)lysine by digestive enzymes. N-2-Propenals
have been identified as the major forms of malondialdehyde in urine. To determine whether available
lysine can be released from the N-2-propenals of lysine in vivo, two preparations containing N-ꢀ-(2-
propenal)lysine and N-R-(2-propenal)lysine or N,N′-di-(2-propenal)lysine were synthesized using
radioactively labeled lysine and were administered to rats by gastric intubation and intraperitoneal
injection. Both preparations were absorbed from the digestive tract, although not as efficiently as
free lysine, but most of the radioactivity was excreted in urine. The radioactive label was also readily
excreted after intraperitoneal injection. It is concluded that the N-2-propenals of lysine are fairly stable
in vivo, so that, although they are absorbed from the gut, most of the absorbed material is not
metabolized and is readily excreted as nonavailable lysine.
KEYWORDS: Malondialdehyde; lysine; N-2-propenals; lipid peroxidation; food proteins; absorption;
bioavailability
INTRODUCTION
Peroxidation of polyunsaturated lipids in foodstuffs during
processing and/or storage leads to losses of available lysine as
determined by chemical and in vivo methods (26-30). In
experiments in which the presence of free and bound forms of
MDA was investigated in enzymatic hydrolysates of foods of
animal origin, the aldehyde was mostly found in the form of
N-ꢀ-(2-propenal)lysine (31) (Figure 1). Therefore, it appears
that the organism is exposed to MDA in the diet mostly in the
form of N-2-propenal derivatives of lysine. N-2-Propenal
derivatives are usually reported as MDA because they are readily
hydrolyzed under the acidic treatment that is required for the
thiobarbituric acid test. Chromatographic separation before
reaction with the thiobarbituric acid reagent is needed to
discriminate between bound and free MDA (9, 32).
The peroxidative decomposition of lipids in foods containing
polyunsaturated fatty acids has detrimental effects on the
nutritional, toxicological, and functional properties of foods (1-
5). Among the products of lipid peroxidation that have been
more extensively studied is malondialdehyde (MDA). This
aldehyde can serve as an index of peroxidative lipid decomposi-
tion and is often analyzed by reaction with thiobarbituric acid
(6-9). MDA readily reacts with functional groups present in
proteins, nucleic acids, and phospholipids, especially amino
groups (10, 11). The main products of the reaction with amino
groups in neutral or acidic aqueous media are N-2-propenals
(12, 13), as well as 4-substituted 1,4-dihydropyridine-3,5-
dicarbaldehyde when alkanals are also present (14, 15). N-2-
Propenals can form Schiff’s bases by reacting with a second
amino group forming N,N′-1-amino-3-iminopropene groups,
which are fluorescent and less stable structures (16). Reactions
with proteins and nucleic acids have been described (12, 17-
22). MDA has been found to be a toxic, carcinogenic, and
mutagenic agent, although initial reports of its damaging effects
might have been overstated to some extent (11, 23-25). The
levels of MDA as measured by reaction with thiobarbituric acid
are increased in pathological conditions such as ischemia-
reperfusion injury, atherosclerosis, and diabetes (reviewed in
(9)).
N-2-Propenals were identified as the main urinary metabolites
of MDA (33), but it remained to be determined whether these
derivatives can be hydrolyzed after absorption from the digestive
tract. N-2-Propenals of lysine in urine do not necessarily
represent the same N-2-propenals that are taken up with the
diet, because N-2-propenals are also endogenously generated
(37). The N-2-mono- (34) and -dipropenal (35) derivatives of
lysine were not hydrolyzed in incubations with liver, kidney,
and intestinal mucosa homogenates, supporting the view that
these derivatives are quite stable in tissues. In the present work,
radioactive labeling of lysine in N-2-propenal derivatives has
been used to assess incorporation into tissues, as well as
excretion, by following the distribution of the radioactive label.
The goal is to determine whether these derivatives are stable or
can be metabolized in vivo, leading to release of lysine. In these
* Corresponding author: Julio Giro´n-Calle, Instituto de la Grasa (Consejo
Superior de Investigaciones Cient´ıficas), Avenida Padre Garc´ıa Tejero 4,
41012 Sevilla, Spain. Phone: (34)954611550. Fax: (34)954616790. E-
mail: jgiron@cica.es.
† Dr. Eduardo Vioque is deceased.
10.1021/jf025681r CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/11/2002

Bioavailability of MDA−Lysine Adducts
J. Agric. Food Chem., Vol. 50, No. 21, 2002 6195
and MDA sodium salt (1.5 mmol) in methanol (20 mM) as previously
described (34). The reaction products were chromatographed on a
Kieselgel 60 column (40 × 1.8 cm, 45 g) using a chloroform/methanol
stepwise elution gradient (3:2, 1:1, 2:3, 1:2, 1:3; 200 mL each per 1.5
mmol of the reagents). The eluted fractions were examined by TLC
using silica gel plates with fluorescent indicator and n-propanol/water
(8:2) as the developing solvent. UV light (254 nm) and 2-thiobarbituric
acid spray reagent (31) were used for visualization of the MDA-
containing lysine derivatives. A chromatographically pure compound
with Rf 0.60 (7% yield) and a mixture of two compounds with Rf 0.47
and 0.37 (22% yield) were obtained. The compound with Rf 0.60 was
N,N′-di-(2-propenal)lysine (DPL) as determined by 13C and 1H nuclear
magnetic resonance: 1H NMR (dimethyl sulfoxide-d6) δ 1.30 (m, 2H),
1.49 (m, 2H), 1.68 (m, 2H), 2.99 (t, 2H enamine), 3.14 (m, 2H imine),
3.58 (t, H), 5.07 (m, 2H enamine), 5.22 (m, 2H imine), 7.30 (m, 2H),
8.80 (m, 2H imine), 8.89 (m, 2H enamine), 7.73 (H amine), 7.90 (H
amine); 13C NMR (dimethyl sulfoxide-d6) δ 22.9, 27.8, 31.3, 43.1, 56.5,
100.0, 158.7, 174.3, 189.6. UV and IR data were also in agreement
with this structure (see below).
The two compounds with lower Rf values were separated by carrying
out a second chromatography of the mixture using a Kieselgel column
as described above. Fractions that afforded single bands of Rf 0.47 and
0.37 on TLC showed identical ultraviolet and infrared spectroscopic
properties, characteristic of the N-2-propenal derivatives of lysine: UV
(ethanol) λ max 280 nm; IR (KBr) ν 3238 cm-1 (Rf 0.47) and 3252
cm-1 (Rf 0.37) (OH, NH), 1601 cm-1 (HN-CdC-CdO), 1406 cm-1
(C-N). The identities of these two compounds, the two N-2-
monopropenals of lysine, N-R-(2-propenal)lysine and N-ꢀ-(2-propenal)-
1
lysine, was established by 13C and H NMR spectroscopy (34). This
separation of the two monopropenals was carried out only for
identification purposes, because they were administered together for
the in vivo experiments.
To obtain radioactively labeled products L-[4,5-3H]lysine monohy-
drochloride was added to the reaction mixtures. Radiochemical purity
was assessed by scanning TLC plates using a Berthold model LB2820-1
linear plate analyzer. The radiochemical purity of the 3H-labeled
preparations that were administered to the experimental animals was
98.5% or better.
Biodistribution Studies. Rats weighing approximately 180 g were
made to fast overnight before the experiments and kept in individual
metabolic cages for urine and feces collection. Gastric intubation of
the samples diluted in 1.5 mL of water was carried out using an 18 G
round-tip needle. The samples were diluted in 0.4 mL of saline solution
for intraperitoneal injection. After anesthesia with diethyl ether, blood
was collected from the heart for plasma preparation, and organs and
tissues were removed. Intestine contents were collected by perfusion
with saline solution. Microsomal fractions of fresh livers were prepared
by differential centrifugation according to Sherr et al. (36, 37).
Samples were prepared for liquid scintillation counting according
to the recommendations of the scintillation liquid manufacturer (Beck-
man, Palo Alto, CA) for the specific type of biological sample. This
included treatment with hydrogen peroxide and acidification with acetic
acid to prevent chemiluminescence. Samples of tissue, feces, plasma,
digestive tract content, and microsomal fractions had to be predigested
using a quaternary base solubilizer (BTS 450 tissue solubilizer). The
accuracy of the liquid scintillation measurements was assured by
repeating the determination after adding an internal standard in selected
samples.
Figure 1. MDA and the N-2-propenals that result from its reaction with
lysine.
experiments, incorporation and excretion of the radioactive label
similar to the incorporation and excretion that is observed after
administration of free lysine would suggest release of lysine
from the administered N-2-propenals. Alternatively, patterns of
incorporation and excretion of radioactivity different from those
corresponding to free lysine would indicate that the N-2-
propenals are not hydrolyzed in vivo and are not a source of
free lysine.
MATERIALS AND METHODS
Materials and Animals. The sodium salt of MDA was prepared as
described by Kikugawa and Ido (14) using 1,1,3,3-tetramethoxypropane
and Dowex 50W-X8 resin obtained from Fluka (Bucks, Switzerland).
MN-Kieselgel 60 (0.063-0.2 mm particle size) for column chroma-
tography and Allugram analytical plates with fluorescent indicator for
thin-layer chromatography were purchased from Macherey and Nagel
(Duren, Germany). Ready Safe liquid scintillation fluid and BTS 450
tissue solubilizer were purchased from Beckman (Palo Alto, CA).
L-[4,5-3H]Lysine monohydrochloride was purchased from Amersham
(Uppsala, Sweden).
Statistics. Data are given as mean ( SEM. One-way analysis of
variance (ANOVA) followed by the Tukey test for several groups was
used to determine significance of differences between groups. A
probability of p > 0.05 was considered significant.
1
13C and H nuclear magnetic resonance spectroscopy was carried
RESULTS
out with Varian XL-200 and Bruker AC 300 instruments using
tetramethylsilane as the internal standard. A Bomem MB-120 instrument
was used for infrared spectroscopy.
Male Wistar rats were obtained from IFFA CREDO (Lyon, France)
and kept in standard conditions for at least 1 week before experiments.
Preparation of Lysine N-2-Propenals. The N-2-propenals of lysine
were prepared by reaction of L-lysine monohydrochloride (1.5 mmol)
Preparation of Lysine Propenals. Reaction of lysine with
MDA in aqueous (13, 38) or methanolic (34) media provides a
mixture of the three possible lysine N-2-propenals, namely,
N,N′-di-(2-propenal)lysine (DPL) and the lysine monopropenals
N-ꢀ-(2-propenal)lysine and N-R-(2-propenal)lysine (MPL) (Fig-
ure 1). For the present study, a preparation of chromatographi-

6196 J. Agric. Food Chem., Vol. 50, No. 21, 2002
Giro´n-Calle et al.
Table 1. Distribution of Radioactivity after Intubation into the Stomach
of Lysine, MPL, or DPLa,b
% of administered dpm
lys
MPL
DPL
urine
feces
11.3 ± 2.0
15.1 ± 2.1
2.7 ± 0.8
30.8 ± 3.4c
26.4 ± 1.0c
0.8 ± 0.6
21.9 ± 5.1
36.5 ± 3.3c
1.0 ± 0.2
digestive content
feces + digestive contents
liver
17.8 ± 1.9
14.91 ± 2.50
7.75 ± 0.62
2.63 ± 0.46
1.48 ± 0.23
1.47 ± 0.10
1.25 ± 0.15
0.72 ± 0.05
0.62 ± 0.02
0.53 ± 0.12
0.38 ± 0.02
31.74 ± 2.6
60.8 ± 4.1
27.2 ± 0.6c
2.14 ± 0.19c
1.05 ± 0.23c
0.36 ± 0.06c
0.26 ± 0.02c
0.29 ± 0.01c
0.15 ± 0.02c
0.20 ± 0.04c
0.16 ± 0.06c
0.13 ± 0.01c
0.08 ± 0.00c
4.81 ± 0.4c
62.8 ± 3.6
37.5 ± 3.4c
0.74 ± 0.17c
0.73 ± 0.12c
0.23 ± 0.03c
0.08 ± 0.02c
0.26 ± 0.06c
0.10 ± 0.01c
0.09 ± 0.03c
0.07 ± 0.01c
0.07 ± 0.03c
0.06 ± 0.01c
2.43 ± 0.1c
61.8 ± 1.6
small intestine
plasmad
kidney
large intestine
stomach
lung
brain
gluteus musclee
heart
Figure 2. Radioactivity in urine collected after intubation into the stomach.
Urine was collected in metabolic cages at three different times after
intubation of the samples into the stomach. Data represent mean ± SEM
(n ) 3). * indicates significant difference (p < 0.05) when compared with
lysine.
tissues total
total
a 12.5 µmol of lysine equivalents of the three 3H-labeled preparations (lysine
and MPL 1.92 × 106 cpm, DPL 5.47 × 106 cpm) were administered. Urine and
feces were collected in metabolic cages for 24 h, after which rats were sacrificed
for tissue sampling. b Values represent mean ± SEM (n ) 3). c Significantly different
(p < 0.05) when compared with lysine. d Values corresponding to total plasma,
assuming 3.5 mL/100 g of body weight. e Values adjusted for 1 g of muscle,
approximate weight of the tissue fragments that were taken.
cally pure DPL and a preparation containing ꢀPL/RPL 7:3
(w/w) (MPL) were obtained by synthesis in methanolic solution
and adsorption chromatography. Spectroscopic data of these
compounds were in accord with what has been previously
described for enaminal-type adducts (13). The 1H NMR analysis
of DPL showed the same trans-enamine-trans-imine tautom-
erism that was previously described for ꢀPL (34, 38) (see
spectroscopic data in Materials and Methods). An apparent
interconversion between the two monopropenals isomers was
observed upon storage of the samples in aqueous or methanolic
solution. Draper and co-workers proposed the formation of an
intermediary cyclic Schiff’s base to explain the same observation
(38, 39).
Figure 3. Distribution of the radioactivity incorporated into tissues.
Radioactivity is expressed as percentage of total radioactivity in tissues
to facilitate comparison among the three samples in different tissues. Data
represent mean ± SEM (n ) 3). All but one of the differences between
lysine and any of the N-2-propenals, or between MPL and DPL, were not
statistically significant (p > 0.05).
Excretion and Tissue Incorporation after Oral Adminis-
tration. To avoid reaction with other components of the diet,
and also to ensure consistent administration of a certain amount
of sample, the N-2-propenals of lysine were administered by
intubation into the stomach instead of by mixing them into the
diet of the animals. The distributions of radioactivity in urine,
feces, and tissues after administration of unaltered lysine, MPL,
and DPL are shown in Table 1 as percentages of administered
counts per minute (cpm). Radioactivity in urine versus time since
administration is shown in Figure 2.
Radioactivity in the urine collected during the first 5 h was
several times higher after administration of MPL or DPL than
after administration of free lysine. More than half of the
radioactivity that was collected in urine after administration of
MPL and DPL was excreted in this period of time (Figure 2).
Elimination of radioactivity in feces was also higher for MPL
and DPL than for lysine (Table 1).
Whereas almost one-third of the radioactivity corresponding
to administered lysine (31.7%) was found in the tissues and
organs that were collected, only 4.8% of the counts correspond-
ing to MPL and 2.4% of those corresponding to DPL were
incorporated into tissues. Accordingly, the bioavailabilities of
lysine contained in these two fractions were estimated to be no
higher than 15% (4.8/31.7 × 100) and 8% (2.4/31.7 × 100),
respectively, of that of the unaltered amino acid. Considering
that the N-2-propenals might have been accumulated in a
nonavailable form in the tissues, e.g., by reaction of the N-2-
propenals with proteins or other components of tissues as such
lysine derivatives, the true availability might be even lower.
The distribution of radioactivity as a percentage of the total
radioactivity incorporated in tissues is shown in Figure 3 to
facilitate comparison among the three samples in different
tissues. Thus, a correction for the different amounts of sample
absorbed from the digestive tract is made. It is apparent that
incorporation into tissues of the absorbed material is essentially
the same for lysine, MPL, and DPL. Only in the large intestine
is a significantly different incorporation of one of the N-2-
propenal samples, DPL, observed.
Incorporation into Liver Microsomes after Intraperitoneal
Injection. To determine regeneration of free lysine available
for protein synthesis, the radioactivity in the hepatic microsomal
fraction of rats injected intraperitoneally with the radiolabeled
samples was determined. Radioactivity in plasma and urine was
determined as well (Table 2). After administration of free lysine,
0.48% of the radioactivity was found in the microsomal fraction,
showing incorporation of the amino acid into newly synthesized
protein, but no radioactivity distinguishable from the background
was found in the microsomal fraction of rats injected with MPL

Bioavailability of MDA−Lysine Adducts
J. Agric. Food Chem., Vol. 50, No. 21, 2002 6197
other hand, it also indicates that not much free MDA, if any at
all, can be released in vivo from this type of lysine derivative.
This might constitute a positive effect of the formation of N-2-
propenals, which, in this way, can be considered as a form of
benign, readily eliminated MDA. All of this information is
consistent with previous research by others and by us concerning
the excretion of bound forms of malondialdehyde (33) and the
stability of the N-2-propenals of lysine in incubations in vitro
with tissue homogenates (34, 35). N-2-Propenals are also formed
by reaction of MDA with nucleic acids. Interestingly, the
reactivity of ꢀPL with nucleic acids was found to be much lower
than the reactivity of free MDA (22).
It remains to be established to what extent the small
incorporation of radioactivity into tissues after oral administra-
tion of the N-2-propenals of lysine represents regeneration of
available lysine. It could also represent reaction of these
derivatives as such with proteins in tissues. Determination of
radioactivity in the hepatic microsomal fraction after intraperi-
toneal administration was carried out to determine whether free
lysine can be released from DPL and MPL. Nevertheless, only
0.48% of the radioactivity administered as lysine was incorpo-
rated in the time frame studied, so that the absence of radio-
activity after administration of DPL or MPL is not statistically
significant for this purpose.
In addition to being formed in foods, MDA is also formed
in vivo, and N-2-propenal derivatives appear to also be products
of the reaction of MDA with proteins in living tissues. This is
supported by the fact that the same kinds of MDA derivatives
mentioned above were found in the urine of fasted rats (38).
Administration of free or bound forms of MDA increases the
amount of these molecules over certain basal levels, considered
to represent endogenously formed MDA (24, 38, 40). In vivo
protein modification by reaction with MDA has also been shown
by immunochemical techniques (10, 42-46). In this context,
our results indicate that the N-2-propenals of lysine are fairly
stable in vivo, suggesting that modification of proteins by
reaction with MDA to form N-2-propenals might be irreversible
and repaired only by substitution of the modified proteins.
Table 2. Radioactivity in Urine, Plasma, and the Hepatic Microsomal
Fraction after Intraperitoneal Injection of Lysine, MPL, or DPLa,b
% of administered dpm
lys
MPL
DPL
microsomesc
plasma
urine
0.48 ± 0.01
2.52 ± 0.20
4.10 ± 2.08
0.02 ± 0.01d,e
0.08 ± 0.01d,e
34.40 ± 7.44d
0.00 ± 0.01d,e
0.05 ± 0.02d,e
20.69 ± 4.78
f
a 6.25 µmol of lysine equivalents of the three 3H-labeled preparations (lysine
and MPL 0.96 × 106 cpm, DPL 2.74 × 106 cpm) were injected, and urine was
collected. After 7 h, rats were sacrificed for liver and plasma extraction. b Values
represent mean ±SEM (n ) 3). c Whole hepatic microsomal fractions counted.
d Significantly different (p < 0.05) when compared with lysine. e Not significantly
different than background (p > 0.05). f Values corresponding to total plasma,
assuming 3.5 mL/100 g body weight.
or DPL. These two samples were readily excreted in urine, and
only residual amounts of radioactivity were found in plasma.
DISCUSSION
Draper and co-workers studied the excretion of bound and
free MDA in rats to which different MDA-containing samples
had been given. Administration of ꢀPL or even free MDA
caused very little excretion of the free aldehyde in urine
(38, 40). Most of it was found as ꢀPL (39) and N-R-acetyl-N-
ꢀ-(2-propenal)lysine. Administration of MDA-treated BSA (38)
or diets promoting lipid peroxidation (32, 41) resulted in
excretion of the same forms of MDA. These studies clearly
established that the N-2-propenals of lysine are the main form
of excretion of MDA, but it still remained to be established
whether these structures can be hydrolyzed in vivo. The fact
that MDA is excreted mostly as N-2-propenals in urine does
not exclude the possibility that N-2-propenals might be hydro-
lyzed in vivo, because the N-2-propenals of MDA can also be
formed endogenously. Thus, N-2-propenals of lysine were found
in the urine of fasted rats (38). The N-2-propenals that are
observed in urine after administration of MDA or related
compounds or diets might originate from the direct excretion
of administered N-2-propenals, or alternatively, they might be
formed endogenously and be derived from hydrolysis of
administered bound MDA.
In the present work, the use of a radioactive label in the N-2-
propenal samples allowed direct tracking of the excretion and
distribution into tissues. Because the lysine moiety carries this
label, regeneration of free lysine by hydrolysis of N-2-propenals
should lead to an incorporation of radioactivity the same way
that administration of the control, free lysine, leads to incorpora-
tion of radioactivity. Our results indicate that no more than 8
and 15% of administered MPL and DPL, respectively, is
converted to free lysine. This implies that transit through the
stomach is not enough to hydrolyze the N-2-propenals ef-
ficiently. Stronger acidic treatment, for instance, hydrolysis in
6 N HCl as carried out for amino acid analysis, is needed for
quantitative release of free lysine (21). Most of the MPL and
DPL that are absorbed from the digestive tract are excreted in
urine, presumably as unaltered N-2-propenals of lysine or in
the form of some other metabolite such as the N-R-acetyl
derivative described by McGirr et al. (38).
ABBREVIATIONS USED
MDA, malondialdehyde; DPL, N,N′-di-(2-propenal)lysine;
ꢀPL, N-ꢀ-(2-propenal)lysine; RPL, N-R-(2-propenal)lysine; dpm,
disintegrations per minute; MPL, lysine N-2-monopropenals.
ACKNOWLEDGMENT
This work was supported by CICYT Grants ALI88-0169,
ALI91-0409, and AGL2001-0526 and by a fellowship from the
Ministerio de Educacio´n y Ciencia (J.G.). We thank Drs. F. J.
Hidalgo and I. Hermos´ın for help with NMR studies and Dr. J.
Vioque for reviewing the manuscript.
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Received for review May 16, 2002. Revised manuscript received July
31, 2002. Accepted August 4, 2002.
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