Identification of acrolein from the ozone oxidation of unsaturated fatty acids

Article abstract of DOI:10.1191/096032799678839527

By-products of lipoperoxidation reactions may be associated with the genesis or the progression of several diseases as arteriosclerosis, diabetes and cancer, among many others. Acrolein, at first a widely distributed environmental pollutant, is currently known as a compound capable of being generated as a result of metabolic reactions within biological systems, highly toxic and the most electrophilic of the α, β-unsaturated aldehydes formed during lipoperoxidation. In the present study: 1. The separation of acrolein and malondialdehyde was achieved at alkaline pH with the use of high voltage capillary electrophoresis in uncoated fused-silica capillaries. 2. It was demonstrated how the oxidation of fatty acids (arachidonic/linoleic) with ozone generates, in dose-dependent form, acrolein as one of the by-products of the lipoperoxidation process. The oxidation of open human erythrocyte membranes with ozone also generated acrolein. 3. After aldolic condensation, aldol-acrolein derivative has a positive reaction with 2-thiobarbituric acid (TBA) and shows a maximum absorption at 498 nm. This novel characteristic is used in its identification after the separation of the by-products. 4. It is possible to suggest that in the classic reaction of the denominated thiobarbituric acid reactive substances (TBARS), when used as an indicator of the degree of peroxidation in biological systems, a portion of acrolein could be present but dwarfed by the TBA-MDA adduct.

Full text of DOI:10.1191/096032799678839527

Human & Experimental Toxicology (1999) 18, 677±682
1999 Stockton Press All rights reserved 0144 ± 5952/99 $15.00
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Identi®cation of acrolein from the ozone
oxidation of unsaturated fatty acids
R Medina-Navarro*,1, E Mercado-Pichardo2, O HernaÂndez-PeÂrez2 and JJ Hicks3
1Specialties Hospital, Medical Research Unit in Biochemistry, Mexican Institute of Social Security, Mexico;
2Metropolitan Autonomous University, Mexico; 3Juarez Mexico City Hospital, Mexico
By-products of lipoperoxidation reactions may be asso-
ciated with the genesis or the progression of several
diseases as arteriosclerosis, diabetes and cancer, among
many others. Acrolein, at ®rst a widely distributed
environmental pollutant, is currently known as a com-
pound capable of being generated as a result of metabolic
reactions within biological systems, highly toxic and the
most electrophilic of the a, b-unsaturated aldehydes
formed during lipoperoxidation. In the present study:
human erythrocyte membranes with ozone also
generated acrolein.
3
4
After aldolic condensation, aldol-acrolein derivative
has a positive reaction with 2-thiobarbituric acid
(TBA) and shows a maximum absorption at 498 nm.
This novel characteristic is used in its identi®cation
after the separation of the by-products.
It is possible to suggest that in the classic reaction of the
denominated thiobarbituric acid reactive substances
(TBARS), when used as an indicator of the degree of
peroxidation in biological systems, a portion of
acrolein could be present but dwarfed by the TBA-
MDA adduct.
1
The separation of acrolein and malondialdehyde was
achieved at alkaline pH with the use of high voltage
capillary electrophoresis in uncoated fused-silica
capillaries.
2
It was demonstrated how the oxidation of fatty acids
(arachidonic/linoleic) with ozone generates, in dose-
dependent form, acrolein as one of the by-products of
the lipoperoxidation process. The oxidation of open
Keywords: acrolein; ozone; malondialdehyde; lipoperoxidation;
capillary electrophoresis
Introduction
For several years it has been known that the
oxidation of lipids or lipo-peroxidation generates a
series of by-products with notable reactivity.
Among these compounds are the aldehydes derived
from peroxidation. Acrolein, 2-propenal or acryl-
aldehyde is the most reactive and cytotoxic
aldehyde identi®ed in peroxidation processes.1 In
such a way that it can form Schiff bases with amino
groups, b-substituted alkenals and addition pro-
ducts, as is the case of the acrolein-lysine adduct
(Na-acetyl-Ne-(3-formyl-3,4 dihydropiperidine) ly-
sine (EDP-lysine), a compound identi®ed through
immuno-histochemical analysis from fatty streak
lesions of arterial tissue in humans.5 Acrolein also
induces a response to stress in alveolar macro-
phages, apoptosis and necrosis.6 In the past it was
known that acrolein could be formed as a metabolic
product from polyamines, from the cytostatic
compound cyclophosphamide, constituting a meta-
bolite of allylic compounds such as methylamine.7
It has been postulated that the cardiovascular
damage observed in experimental animals and
produced by allylamine is mediated by the forma-
tion of acrolein through enzymatic deamination
catalyzed by the enzyme semicarbazide sensitive
amino-oxidase (SSAO).8 ± 10 The assessment of acro-
lein in atmospheric air is generally veri®ed using
gas chromatography.11 Given the instability, in the
case of endogenous acrolein, it is best to identify the
adducts formed in its interaction with amino acids
and its separation using reverse-phase HPLC5 such
as has been done with other derived aldehydes, as is
the case of 4-hydroxy-2-nonenal.12 The identi®ca-
tion and measuring of aldehydes and their metabo-
lites are good indicators of lipidic peroxidation. The
most widely used lipoperoxidation index is without
the past, it was known as
a component of
environmental pollution and recognised for its
irritating power and its ability for depressing the
respiratory immune response.2 It is now known as
an important lipoperoxidation product. Immunoen-
zymatic assays used for detecting acrolein have
revealed that a considerable amount of the aldehyde
is freed during the oxidation of low density
lipoproteins (LDL) when exposed to Cu2+ ions.3 It
was recently shown that during the respiratory
burst of activated neutrophils, the hydrogen-chlor-
ide peroxide myeloperoxidase system can convert
hydroxi-amino acids into unsaturated a, b alde-
hydes, among them acrolein, in large amounts.4
Acrolein could be implicated in the etiopathology
of arteriosclerosis. Its highly electrophilic character
allows it to avidly react with several amino acids, in
*Correspondence: R Medina-Navarro, P.O. Box A-055, Coahuila
No 5, Colonia Roma, MeÂxico D.F. MeÂxico, C.P. 06703
Received May 1999; revised August 1999; accepted 7 September
1999
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Identification of acrolein
R Medina-Navarro et al
678
a doubt, the formation of malondialdehyde (MDA)
where this aldehyde reacts with 2-thiobarbituric
acid (TBA) at a low pH and high temperature for a
coloured reaction with a strong absorption of
approximately 532 nm.13
rationale behind this was that the ®rst fraction
would contain the ®rst compound given that it
includes the volume equivalent to the distance to
the window detector, that is to say 7 cm. Given that
the concentration of the ®rst compound was
expected to be 9.5 times lower, ®ve injections of
the same fraction were done for each. The volumes
were adjusted at the end. The fraction was collected
in micro-tubes containing 0.375% TBA solution.
Methods
The following reagents used were purchased from
Sigma Chemical Co., USA, and were of the
maximum available purity: 1, 1, 3, 3-tetramethoxy-
propane (malondialdehyde bis (dimethyl acetal),
acrolein 90% (GC), 9, 12-octadecadienoic acid
(linoleic acid) and 5, 8, 11, 14-eicosatetraenoic acid
(arachidonic acid). The capillary regenerator solu-
tion and the capillaries used (75 mm657 cm) were
purchased from Beckman Instruments, Inc. (Full-
erton, CA, USA). The HPLC grade water was
purchased from Mallinckrodt Baker, S.A. de C.V.
(Xalostoc, Mexico).
Fatty acid vesicles (ARLI)
Precisely 40 mg of linoleic acid and 0.40 mg of
arachidonic acid were mixed in 4 ml of Tris/HCl at
pH 8.0. A suspension was formed by intense
agitation for a period of 30 min. Later on, the
mixture was taken to a ®nal concentration of
7.2 mM using the same buffer.14 Vesicles were
always freshly prepared. Immediately before the
assays, 200 ml of the ARLI solution were taken and
the volume adjusted to 1000 ml with HPLC water for
a ®nal concentration of 1.44 mM. The resulting
solution was suctioned into a plastic syringe where
the oxygen-ozone mixture was used for verifying
the reaction. The ®nal intense agitation for 20 min
insures the complete exhaustion of the ozone
oxidising capacity. The polar phase containing the
aldehydes is obtained after the lipid extraction with
chloroform-methanol (2 : 1), agitated for 10 min and
centrifuging the sample at 2500 g for 15 min.
System and separation parameters
The samples were analyzed using the capillary
electrophoresis system P/ACE TM series 5000 from
Beckman Instruments, Inc. (Fullerton, CA, USA)
online with a Diode Arrangement Detector for
spectral analysis at a wavelength range of 190 to
600 nm (512 diodes). In all cases, the free capillary
columns measuring 57 cm long (50 cm to the
detection window) and 75 mm of internal diameter,
built within the P/ACE cartridge for capillary
cooling, were used. The samples were pressure-
injected at 0.5 p.s.i. for 5 s. The optimum separation
voltage used in all cases was 25 kV (normal
polarity) and the treatment between capillary runs
was: 4 min of capillary washing with the running
buffer; 4 min with the 1.0% NaOH regenerating
solution; 6 min washing with water and 6 min of
draining previous to the injection of the sample
with the running buffer (high pressure: 15 p.s.i.). In
all cases, the running buffer used was 0.1 M borate
pH 8.27. For the analysis, the Beckman system Gold
software # 1991 was used.
Erythrocyte ghosts
Open erythrocyte membranes were prepared by
hypotonic lysis obtained from complete blood
samples from adult volunteers, collected in sterile
test tubes containing heparin as an anticoagulant, as
originally described by Steck.15 The entire process
was veri®ed at 48C and on the same day the
experiment was performed. Finally, the membra-
ne's protein concentration was 3.5 mg/ml on the
average. The oxidation takes place using a volume
equivalent to 70 mg and adjusting exactly to 1000 ml
with HPLC water. The same procedure as described
for ARLI including the extraction with the chloro-
form-methanol 2 : 1 mixture was followed.
Fractions obtention
Exposure to ozone
The total estimated capillary volume at 278C and
0.79861072 pois was 2544 nl. Taking into account
that the ®rst separated compound appeared at
approximately 3 min after having started the run,
the system was programmed to stop the separation
after 3 min of the run. The system was ordered to
inject 312.4 nl of the mixture at low grade pressure
(0.5 p.s.i.) for 1 min. This sequence is repeated 50
times and corresponds to the ®rst fraction.
Every ®ve runs, the apparatus was programmed
to carry out another four consecutive injections (in
their corresponding tubes) equivalent to additional
four fractions containing 312.4 nl each. The main
The ozone used in the experiments was generated
from a high voltage electric arch (Biozon, Fagor;
Bask Tec, Mexico), connected to an ultrapure
oxygen tank. The ozone mixture was deposited in
a container where the mixture circulated con-
stantly. The mixture volumes were extracted using
a needle and a syringe, through a rubber stopper
adhered to the container. The syringes were sealed
and all the reactions were carried out inside,
agitating the mixture for 30 min using a magnetic
stirrer. Ozone concentration curves were carried
out within the plastic syringes each time an
experiment was carried out measuring the iodine
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Identification of acrolein
R Medina-Navarro et al
679
concentration (e=25 200/350 nm) as a product of
the stechiometric reaction of ozone with the Kl.16,17
The ozone concentration estimated was 1 mg per ml
of the gas mixture taken from the container.
aqueous phase of a mixture that shows the same
absorption maximum at 266 nm. The electrophore-
tic separation reveals the formation of two peaks
with the same migration times as acrolein and
MDA. Finally, when separating the fractions, it is
shown that their reaction with TBA corresponds to
the absorbencies of both acrolein (498 nm) and
MDA (532 nm) standards in its reaction with TBA,
demonstrating that the ozone oxidation of unsatu-
Preparation of standards
A malondialdehyde (MDA) stock solution was
prepared from the acid hydrolysis of the compound,
previously distilled malondialdehyde-bisdimethy-
lacetal.18 Approximately 164.2 mg of the compound
were removed and 100 ml of 1% sulphuric acid (v/
v) were added. After letting rest at room tempera-
ture for 2 h, 1 ml of the solution was taken and
adjusted to a 100 ml volume with more 1%
sulphuric acid (v/v). The stock solution has an
approximate concentration of 10 mM, while the
®nal solution for 0.1 mM spectrophotometric assess-
ment, veri®ed using an e=13 700 and e=153 000 for
the MDA-TBA adduct, should have a variation of no
more than 2%. Approximately 100 ml of this
solution was taken and the same volume of 1%
NaOH added. The solution was allowed to rest in
the dark for 30 min. The formation of the NaMDA
form is veri®ed at 266 ± 267 nm.19 The volume is
adjusted to 1 ml using a 0.01 mM pH 8.27 borate
buffer. The ®nal concentration of the solution was
10 mM. In acrolein's case, at 28C, 20 ml of the bottle
were removed (90%, d=0.849) and dissolved in
20 ml of water (HPLC grade). From this solution,
100 ml were taken to which an equal volume of 1%
NaOH was added. The aldolic condensation was
monitored at 266 nm. The ®nal acrolein solution
was 1.6 mM. Under these conditions, additional
spectral readings and electrophoretic runs were
conducted. The uv/vis scannings were carried out
with a spectrophotometer Beckman DU-650, Beck-
man Instruments, Inc. (Fullerton, CA, USA).
rated fatty acids can generate acrolein as
peroxidation by-product.
a
The oxidation of unsaturated fatty acids (arachi-
donic/linoleic) by ozone generates a hydrosoluble
mixture with strong absorption at 266 nm at an
alkaline pH (Figure 1). The absorption of the
mixture obtained is proportional to the degree of
oxidation, that is, to the concentration of ozone. In
the inserted graph, the same maximum absorbency
at 266 nm is shown after oxidising the open human
erythrocyte membranes with ozone. The mixture of
components obtained after the separation of lipids
with organic solvents shows a positive reaction
with TBA, with the characteristic coloration of what
are known as TBARS. The separation with high
voltage, alkaline pH and in an open capillary of
75 mm of internal diameter of the mixture before-
mentioned, reveals two peaks with retention times
equivalent to 2.7+0.25 and 5.8+0.23 min (Figure
2). As a result of the analysis of several aldehydes, it
was proven that the retention (or migration) times
correspond to acrolein and MDA respectively, as
was proven when testing the standards of these
aldehydes under the same separation conditions
The values reported of the migration times are
expressed as mean+standard deviation, while the
percentage dispersion values correspond to varia-
tion coef®cients.
Results
In the present study, we observed that the product
obtained from the aldolic condensation of an
acrolein standard presents a positive reaction with
TBA and shows a maximum absorption at 498 nm.
Under the same conditions,
a malonaldehyde
standard presents the maximum characteristic of
the MDA:TBA conjugate 1 : 2 at 532 nm.20 However,
the ultraviolet absorption of both compounds shows
the same maximum at 266 nm at pH 8.2. An
acrolein and malonaldehyde mixture at an alkaline
pH was separated by capillary zone electrophoresis
(free solution capillary electrophoresis). The ozone
oxidation of pure fatty acids (arachidonic/linoleic)
in a suspension gives as a result, the freeing in the
Figure
1
Increase in the absorption at 266 nm of a polar
fraction extracted from the mixture of fatty acids (arachidonic/
linoleic) or ARLI, exposed to 1.37 (a), 2.75 (b), 4.13 (c), 5.51 (d),
6.89 (e) mg of ozone/nmol of fatty acids. The exposure took place
within the plastic syringes from which the ozone aliquots were
taken as described in the Methods section. In the inserted graph,
the polar phase of a sample of open erythrocyte membranes
oxidised with 3.8 g of ozone/mg of membrane protein is shown
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Identification of acrolein
R Medina-Navarro et al
680
(Figure 3). The inserted ®gure shows how the
independent reactions from TBA with acrolein
and MDA presented close, but yet different absorp-
tion peaks (acrolein at 498 nm and MDA at 532 nm).
However, it is clear that only 5% (calculated area
%) of acrolein is generated from the total mixture
reactive to TBA.
In Figure 4, the electropherograms corresponding
to the product of the oxidation of erythrocyte
membranes (discontinuous line) and an aldol-
acrolein standard (continuous line) to 266 nm are
shown. An identical migration time of 2.7 min is
shown. The variation between two consecutive runs
was less than 1.5%. When the capillary conditions
are optimum, the migration is not considerably
affected after 20 min. Once there is capillary
instability, it may be re-established with the
following treatment: water for 30 min; HCl 1 M for
5 min; water for another 30 min and capillary
regeneration with NaOH 1% for 5 min. The max-
Figure 2 Electropherogram of the oxidation products from fatty
acids (araquidonic/linoleic) after the extraction with organic
solvents. The software analysis of the area % reveals two peaks
correspondent to 5 and 95% approximately. The separation
conditions were the same as those described for the standards in
Figure 3. The ozone concentration was 4.2 mg/nmol of free fatty
acids. The maximum variation estimated for the migration times
of ten samples was 9.3 and 4.1% for acrolein and malondialde-
hyde, respectively
Figure
4
Superpositioning of two electropherograms corre-
sponding to an acrolein standard and the peak corresponding
to the product of the oxidation of erythrocyte membranes with
ozone. The migration time of the two peaks was equal or very
proximate between the two successive assays.
electropherogram is shown
A selected
Figure
5
Spectral scan of the fractions separated from a
hydrosoluble phase of erythrocyte membranes after being
oxidised with ozone. The TBA reaction took place and a sample
was taken without being submitted to the same conditions. The
maximums of the fractions correspond to acrolein (498 nm) and
malondialdehyde (532 nm). The absorbencies were found to be
within the limits of the spectrophotometer. The ozone concen-
tration in this case was 10 cm3, equivalent to 9 mg of ozone/nmol
of free fatty acids. The separation procedure is described in the
Methods section
Figure 3 Electropherograms of the acrolein and malondialde-
hyde standards. Conditions: open capillary of fused-silica, 75 mm
of internal diameter650 cm of distance from the detector;
potential applied of 20 Kv/40 mA; borate buffer 0.1 M pH 8.27
at a stable temperature of 278C. The variation between the two
consecutive runs was less than 1.5%. In the inserted graph,
spectrum runs of the products of the reactions of the acrolein
and MDA standards with the TBA
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Identification of acrolein
R Medina-Navarro et al
681
onaldehyde reacts with TBA at an acid pH.14 In the
experiments reported herein, we verify that acrolein
positively reacts with TBA, which translates in a
strong absorption at 498 nm. This reaction can take
place after aldolic condensation. Taking into
account that only a scarce amount of acrolein is
generated from a mixture of fatty acids after being
oxidised, it is possible that in the classical detection
of malonaldehyde using the thiobarbituric acid
reaction, a small fraction of TBA-acrolein could
exist, eclipsed by the presence of the TBA-MDA
adduct in large amounts (Figure 6). An important
implication of this fact is that between a,b-
unsaturated aldehydes, acrolein is the strongest
electrophilic and shows the highest reactivity with
sulphydryl groups of cysteine, imidazole groups of
histidine and amino groups of lysine, among others,
even superior to the reactivity of 4-hydroxyl-2-
nonenal.5,23
Figure 6 Explanation in the text
The percentage area of the fraction analyzed by
electrophoresis revealed that a portion of approxi-
mately 5% of the ozone oxidation product corre-
sponds to acrolein, while the other 95% is MDA.
Given that the formation of many other compounds
during the intense oxidation of unsaturated fatty
acids has been proven,1,14 it is clear that the
separation conditions and the detector used dis-
criminate the presence of other possible peaks in
the mixture. In this sense, the diode arrangement
allows to establish that only a sign is seen around
266 nm with a bandwidth of 10.
imum variation estimated for the migration times of
ten samples was 9.3% and 4.1% for acrolein and
MDA, respectively.
The separation of the fractions of an oxidised
sample of erythrocyte ghosts with ozone, after
reaction with TBA, shows the formation of two
spectrophotometric images (Figure 5) with max-
imum absorbency at 498 nm and 532 nm (acroline
and MDA, respectively).
Finally, the principle of the separation in the
zonal capillary electrophoresis mode is the differ-
ence in the size of the solutes and its charge at a
speci®c pH. In the present case, the condensation of
aldehydes veri®ed at an alkaline pH is a key factor
that insures its detection. The presence of hydroxyl
groups (that modify the molecular polarity and
therefore, migration), plus an increase in the size of
the molecule due to the corresponding condensa-
tion, could be the two main factors contributing to
the separation, together with the resolution ability
of the high voltage capillary electrophoresis system.
Discussion
Acrolein is a highly toxic compound. Concentra-
tions of 0.21 p.p.m. in the air are able to cause
important in¯ammatory changes in the lungs, liver,
kidneys and brain of experimental animals. In
humans, it has caused deaths with concentrations
under 10 p.p.m.21,22 In addition, it seems clear that
acrolein is implicated in the genesis and progres-
sion of many vascular lesions, including those
associated to arteriosclerosis.3 ± 5 It is known that
acrolein may come from different sources, among
them the combustion generated from the burning of
fossil combustion and ®res, cigarette smoke, etc. It
is also known that it can come about endogenously
as a metabolite of several compounds, among them
allylic alcohol, allylamine and the anti-cancer
agent, cyclophosphamide.7,8 In the current study, it
was shown that the oxidation of unsaturated fatty
acids and erythrocyte membranes with ozone can
generate acrolein. In spite of the fact that the
ozone's main toxic effect is related to its oxidising
action, the information presented is direct evidence
of another possible enhanced hazardous effect of
ozone.
Acknowledgements
The authors gratefully acknowledge the Consejo
Nacional de Ciencia y Tecnologia (CONACYT) and
the Sistema Nacional de Investigadores (SNI), MeÂx-
ico City for their financial assistance and general
support.
It has been said in the past that from carbonylic
compounds derived from peroxidation, only mal-
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Identification of acrolein
R Medina-Navarro et al
682
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