Evidence for hydroxyl radical generation during lipid (linoleate) peroxidation

Article abstract of DOI:10.1021/ja801858e

The autoxidation of methyl linoleate in benzene at 37 °C by peroxyl radicals was found to generate hydroxyl radicals (?OH) from a secondary oxidation mechanism. The yield of hydroxyl radicals (～2%) was determined by trapping these reactive radicals with benzene to give phenol. We propose that αC-H hydrogen abstraction from lipid hydroperoxides, the main autoxidaion products, is the source of hydroxyl radicals. Copyright

Full text of DOI:10.1021/ja801858e

Published on Web 07/03/2008
Evidence for Hydroxyl Radical Generation During Lipid (Linoleate)
Peroxidation
Mathieu Frenette and Juan C. Scaiano*
Department of Chemistry, UniVersity of Ottawa, Ottawa, Ontario K1N 6N5, Canada
Received March 12, 2008; E-mail: tito@photo.chem.uottawa.ca
Scheme 2. Proposed Mechanism for the Oxidation of LOOH by
LOO• to Form a Ketone (L)O) and a Hydroxyl Radical
Reactive oxygen species (ROS) are collectively responsible for
most oxidative stress occurring in living organisms, and hydroxyl
radicals (HO•) are the most reactive of all ROS. Some observed
oxidation products appear to be derived from reactions involving
HO•, and therefore, mechanisms for the formation of hydroxyl
radicals in vivo have been the subject of research interest for many
decades. Presumably, the most abundant source of HO• in vivo is
the Fe- and Cu-catalyzed Fenton decomposition of hydrogen
peroxide.1
In this communication, we present strong experimental evidence
that hydroxyl radicals are also formed during lipid peroxidation, a
very common process associated with oxidative stress. Polyunsatu-
rated lipids are prime targets for free radical induced oxidation.
The primary oxidation products are mostly hydroperoxides (LOOH,
Scheme 1), and the mechanisms involved in their formation are
well-established.2
very exothermic since the breaking of the weak peroxide bond is
energetically compensated by ketone formation. Reactions of this
type were proposed to explain the formation of ketones in the high-
temperature autoxidation of alkanes5 and, more recently, to explain
the formation of acetophenone during the autoxidation of ethyl-
benzene.6 In theory, this mechanism could explain the formation
of hydroxyl radicals from any secondary (or primary) hydroperoxide.
To explore the viability of this reaction in lipid peroxidation,
we studied the oxidation of methyl linoleate (LH) initiated by the
free radical initiator 2,2′-azo-bis-isobutyronitrile (AIBN). The
reactions were carried out in the dark at 37 °C under air in benzene.
If hydroxyl radicals are formed, they will react with benzene to
yield phenol.7 The generation of phenol from benzene is a selective
test for HO• since the control experiment (with no oxidizable lipid,
but with a peroxyl radical source) did not yield significant amounts
of phenol (Figure 1, trace “CONTROL”).8 In Figure 1, we plot the
measured phenol concentration (GC/MS) as the oxidation of methyl
linoleate in benzene progresses. While the yields of phenol are
modest, these results are strong evidence that HO• is formed during
lipid peroxidation. We propose that the source of HO• is from the
reaction of LOO• with LOOH according to Scheme 2. The upward
curvature for the formation of phenol also supports a secondary
oxidation as its precursor.
Scheme 1a
a A: General lipid peroxidation mechanism. B: Mechanism as it applies
to polyunsaturated fatty acids;2b kinetic products shown, other minor isomers
also expected. Only propagation steps are shown. Methyl linoleate has R,R′
) CH3(CH2)4 and CH3OCO(CH2)7.
If free radical oxidation of lipids is allowed to continue in the
absence of chain-breaking antioxidants, ketones and aldehydes are
formed and these molecules are known to be biologically active.3
Despite significant interest in lipid peroxidation, the mechanism
for the formation of lipid ketones (also called oxodienes) has eluded
a satisfactory explanation for many years.4 In this contribution, we
provide evidence for a simple mechanism that forms ketones from
the corresponding lipid hydroperoxides (Scheme 2). Notably, the
mechanism provides a pathway in which hydroxyl radicals are
formed during metal-independent lipid peroxidation.
We note that the most likely reaction of peroxyl radicals with
hydroperoxides is the abstraction of the hydrogen atom from the
LOO-H bond;3b however, this is effectively an identity reaction,
which at most exchanges one peroxyl radical for another.
At first glance, the generation of HO• from a lipid peroxyl radical
(LOO•) may seem unlikely, but the O-O cleavage of Scheme 2 is
Figure 1. Phenol is formed during the 37 °C autoxidation of methyl
linoleate (LH) under air in benzene due to HO• formation. Experimental
conditions: (A) 0.372 M LH, 0.0189 M AIBN; (B) 0.189 M LH, 0.0189 M
AIBN; (CONTROL) 0 M LH, 0.0189 M AIBN.
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9634 J. AM. CHEM. SOC. 2008, 130, 9634–9635
10.1021/ja801858e CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
We also turned to DFT calculations9 in an attempt to establish
if computed thermodynamic energies were consistent with the
mechanism proposed. We set out to compare the H-donating ability
of the unsaturated lipids (LH) and the corresponding lipid hydro-
peroxide (LOOH). The activation energies for reaction between a
model peroxyl radical (MeOO•) and LOOH and LH moieties were
calculated (see Supporting Information). While the secondary
reaction has a slightly higher transition state free energy,12 the
release of a hydroxyl radical remains Very thermodynamically
favorable with an exergonicity (∆G) of -42.2 kcal/mol.
Figure 2. Normalized GC-MS signals for m/z ) 310 and m/z ) 308,
corresponding to LOOH (major peak only, after Ph3P reduction) and to
L)O (major peak). The experimental conditions are identical to trace B in
Figure 1; note the similar upward curvature for L)O and phenol formation.
In summary, we have obtained evidence that RC-H hydrogen
abstraction from hydroperoxides formed during lipid autoxidation
can lead to the formation of ketones and hydroxyl radicals in a
long overlooked path to these intermediates.
Acknowledgment. We acknowledge generous financial support
from the Natural Sciences and Engineering Research Council of
Canada. M.F. is grateful for scholarships from OGS and the
“Fondation Baxter & Alma Ricard”. Thanks are due to Dr. Chad
Beddie for assistance with DFT calculations, Dr. Keith U. Ingold
for valuable discussions, and to Dr. L. Ross C. Barclay for the
very generous gift of an oxygen uptake apparatus. Some calculations
were performed on the Shared Hierarchical Academic Research
Computing Network (SHARCNET: www.sharcnet.ca).
Figure 3. Oxygen consumed in function of time plotted as a fraction of
oxygen consumed per LH in the system, with linear fit (same conditions as
Figure 1B). See Supporting Information for more information.
Supporting Information Available: Full experimental and theoreti-
cal details; complete ref 11. This material is available free of charge
via the Internet at http://pubs.acs.org.
We also monitored by GC-MS the formation of the major
hydroperoxide peak (LOOH, m/z ) 310 after reduction to the
alcohol with Ph3P) and the major ketone peak (L)O, m/z)308).
The growth of L)O and phenol both shows a similar upward
curvature, but LOOH shows a slight downward curvature (Figure
2). This supports the mechanism proposed in Scheme 2. The
difference between phenol and LOOH also appeases concerns that
unreduced hydroperoxides could generate hydroxyl radicals in the
GC/MS injector and would account for the phenol formed.
We also monitored the oxygen uptake during the autoxidation
of LH (Figure 3). Under these conditions, we estimate the yield of
hydroxyl radical per oxygen molecule consumed to be ∼1% after
50 h, corresponding effectively to a 2% reaction yield, given the
stoichiometric need for two oxygen molecules to produce one HO•
radical (see Schemes 1 and 2). This yield is calculated for a level
of oxidation well above naturally occurring systems; however, we
think the proposed reaction is more probable in membranes due to
the geminate co-localization of both reagents; in other words, LOOH
and LOO• are “born” together in the lipid membrane. More
mechanisms could be imagined for the formation of hydroxyl
radicals from this complex reaction mixture, but we believe the
mechanism shown in Scheme 2 is valid for many reasons, some
mentioned above. Preliminary data on the autoxidation of methyl
oleate (only one cis double bond) in benzene under air also show
the characteristic upward curvature for phenol and the lipid ketone
products (data not shown). Comparison of the data in Figures 1
and 3 suggests that abstraction from LH is about an order of
magnitude faster than from LOOH (see Supporting Information).
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kcal/mol (see Supporting Information for details).
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