Full text of DOI:10.1002/1438-9312(200110)103:10<683::AID-EJLT683>3.0.CO;2-T

Eur. J. Lipid Sci. Technol. 103 (2001) 683–687
683
Marjukka Mäkinen,
Marja Kähkönen,
Anu Hopia
Ascorbic acid and ascorbyl palmitate have only
minor effect on the formation and decomposition
of methyl linoleate hydroperoxides
Department of Applied
Chemistry and Microbiology,
Food Chemistry Division,
University of Helsinki,
Finland
Effects of ascorbic acid (AA) and ascorbyl palmitate (AP) on lipid hydroperoxides were
evaluated during the formation and decomposition of methyl linoleate hydroperoxides
(ML-OOH). AA and AP at 1 and 10 mM levels had no effect on the formation of ML-
OOH during the autoxidation of methyl linoleate at 40 °C. However, depending on the
reaction medium, AA and AP at 0.2 and 2 mM either slightly inhibited or accelerated
the decomposition of 40 mM cis,trans ML-OOH in hexadecane or in hexadecane-in-
water emulsion. The increased decomposition rate of ML-OOH, when compared to a
control sample, was apparently due to the reductive activity of AA and AP on metal ions
present in the system, as the addition of EDTA improved the stability of ML-OOH. The
more detailed analysis of the decomposition reactions of ML-OOH suggests that under
favorable reaction conditions AA and AP were, to some extent, capable of acting as hy-
drogen atom donors to peroxyl radicals and reducers of hydroperoxides to more stable
hydroxy compounds. However, since all these effects of AA and AP on lipid hydroper-
oxides were relatively small, it is assumed that the antioxidative activity of AA and AP
as well as their effect on the stability and reactions of lipid hydroperoxides in biological
systems and in foods is mainly related to their synergistic interactions with other an-
tioxidative compounds such as tocopherols.
Keywords: Antioxidants, ascorbic acid, ascorbyl palmitate, lipid hydroperoxides,
oxidation.
1
Introduction
as exemplified by methyl linoleate hydroperoxides. Each
tocopherol showed different characteristics in its speed to
Ascorbic acid (AA) and ascorbyl palmitate (AP) are im-
portant antioxidants in biological systems and in foods.
They are excellent scavengers of aqueous free radicals
and act synergistically with tocopherols by regenerating
tocopheroxyl radicals [1–2]. However, the reported effects
of AA and AP on lipid hydroperoxides are controversial.
Investigations with results suggesting AA and AP as in-
hibitors or accelerators of hydroperoxide formation and
decomposition have been reported [3-9]. Especially the
ability of AA and AP to accelerate the hydroperoxide de-
composition in the presence of metal ions has attracted
much attention in the literature [10–13] since it is accom-
panied by the re-initiation of new oxidation chain reac-
tions.
donate hydrogen atoms to peroxyl radicals and thus to in-
hibit the isomerization of cis,trans to trans,trans methyl
linoleate hydroperoxides. Assuming that also AA and AP
are chain-breaking antioxidants, they should be capable
of acting as hydrogen atom donors to peroxyl radicals and
thus to inhibit the free radical reactions. According to
Marinova and Yanishlieva [5] AP is a radical scavenger of
peroxyl radicals in the oxidizing lipid systems. In contrast,
Porter [16] and Cillard and Cillard [3] suggested that AA
can not donate hydrogen atoms to lipid peroxyl radicals
during the autoxidation process. Other possible mecha-
nisms responsible for the effects of AA and AP on lipid hy-
droperoxides include the reduction of hydroperoxides to
more stable hydroxy compounds [17-18] and the interac-
tions of AA and AP with metal ions [9–13]. However, the
knowledge of the effects of AA and AP on lipid hydroper-
oxides is still insufficient.
Under mild reaction conditions the formation and decom-
position of lipid hydroperoxides proceed through free rad-
ical chain reactions. In our previous studies [6, 14–15] we
showed that α-, γ- and δ-tocopherols efficiently inhibited
both the formation and decomposition of hydroperoxides
In the present study the effects of AA and AP on lipid hy-
droperoxides were evaluated both during the formation
and decomposition of methyl linoleate hydroperoxides
(
ML-OOH). The formation of ML-OOH was followed dur-
Correspondence: Marjukka Mäkinen, Department of Applied
Chemistry and Microbiology, Food Chemistry Division, P.O. Box
ing the autoxidation of methyl linoleate in a bulk system.
For decomposition studies, cis,trans ML-OOH were dilut-
ed either to hexadecane or to hexadecane-in-water emul-
sion. In the emulsion model, EDTA was added as a metal
2
5
7, 00014 University of Helsinki, Finland. Phone: +358-9-191-
8228, Fax: +358-9-191-58475; e-mail: Marjukka.Makinen@
Helsinki.fi
©
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
0931-5985/2001/1010-0683 $17.50+.50/0

684
Mäkinen et al.
Eur. J. Lipid Sci. Technol. 103 (2001) 683–687
chelator. The ratio of cis,trans to trans,trans hydroperox-
ides as well as the formation of hydroxy compounds were
followed during the decomposition. Thus further insight
on the effects of AA and AP on the intermediate radicals
and the reaction sequences of hydroperoxide decomposi-
tion was gained.
ed (U50 Control Ultrasonic Processor, IKA Labortechnik,
Germany) at high frequency (30 kHz) for 2 min in an ice
bath. Emulsion samples (10 ml) were incubated at 40 °C
in the dark in screw-capped glass vials (20 ml) with mag-
netic stirring. All the samples were prepared and analyzed
as duplicates.
2
Materials and methods
2.5 HPLC analysis
2
.1 Materials
Decomposition of ML-OOH was followed by normal-
phase high-performance liquid chromatography as
described before [6, 14]. Four hydroperoxide isomers,
Methyl linoleate was purchased from Nu-Check Prep Inc.
(Elysian, MN, USA). Ascorbic acid was from Merck
(Darmstadt, Germany, purity >95%). Ascorbyl palmitate
(purity >95%), hexadecane (purity >99%), Tween 20
(polyoxyethylene sorbitan monolaurate) and EDTA (ethyl-
13-cis,trans ML-OOH, 13-trans,trans ML-OOH, 9-cis,trans
ML-OOH and 9-trans,trans ML-OOH were separated by
HPLC. The HPLC method also enabled the simultaneous
analysis of the four methyl linoleate hydroxy compounds
linoleate formed during decomposition.
enediamine tetraacetic acid, purity >99%) were from
Sigma Chemicals Co. (St. Louis, MO, USA). Other sol-
vents were purchased from Rathburn (Walkerburn, Scot-
land, HPLC grade).
3
Results and discussion
Ascorbic acid and ascorbyl palmitate at 1 and 10 mM
levels neither inhibited nor accelerated the formation of
ML-OOH during the autoxidation of methyl linoleate at
2
.2 Autoxidation of methyl linoleate
Methyl linoleate samples (1.4 g) were oxidized in the
dark at 40 °C in glass vials (8 ml) with magnetic stirring.
AA and AP were added at levels of 1 and 10 mM. Sample
aliquots were taken in regular intervals and investigated
with high-performance liquid chromatography (HPLC)
analysis. Samples were prepared and analyzed as dupli-
cates.
40 °C (Fig. 1). This result suggests that AA and AP alone
are not capable of inhibiting the propagated reactions
of lipid autoxidation in bulk systems. The antioxidative
effects of AA and AP in foods and in biological systems
are most likely related to the synergistic interactions of
AA and AP with other antioxidants such as tocopherols.
AA and AP are capable of regenerating tocopheroxyl
radicals formed in the reactions of tocopherols with lipid
radicals. Several groups have demonstrated the impor-
tance of this reaction [1, 16, 21–22].
2.3 Decomposition of cis,trans ML-OOH
in hexadecane
Methyl linoleate was oxidized in the dark with 5% α-toco-
pherol to produce only cis,trans ML-OOH [6, 19].
Cis,trans ML-OOH was separated from non-oxidized
methyl linoleate as described before [6]. The purified
cis,trans ML-OOH was diluted in hexadecane to give a
The decomposition rate of ML-OOH without added AA
and AP was approximately the same in hexadecane as in
40 mM solution. AA and AP were added as freshly pre-
pared methanol solutions and methanol was evaporated
from the samples under nitrogen, to give final concentra-
tions of 0.2 and 2 mM. ML-OOH samples (3 ml) were
incubated at 40 °C in dark in screw-capped glass vials
(12 ml) with magnetic stirring. All the samples were pre-
pared and analyzed as duplicates.
2.4 Decomposition of cis,trans ML-OOH
in emulsion
From 40 mM cis,trans ML-OOH in hexadecane, a 10%
hexadecane-in-water emulsion was prepared [4, 20]. The
amount of AA and AP were the same as above in the bulk
hexadecane, namely 0.2 mM and 2 mM in hexadecane.
EDTA was added to a 50 µM level. Emulsions were made
with 1 g hexadecane and distilled Milli-Q water with 0.1 g
of Tween 20 as emulsifier. Emulsions were then sonicat-
Fig. 1. Effect of ascorbic acid and ascorbyl palmitate on
the formation of ML-OOH during the autoxidation of
methyl linoleate at 40 °C. Control ( ), 1 mM ascorbic acid
♦
(ꢀ), 10 mM ascorbic acid (ꢀ), 1 mM ascorbyl palmitate
(ꢀ) and 10 mM ascorbyl palmitate (ꢀ).

Eur. J. Lipid Sci. Technol. 103 (2001) 683–687
Action of ascorbic acid and ascorbyl palmitate
685
Tab. 1. Effects of ascorbic acid (AA) and ascorbyl palmi-
tate (AP) on the induction period for the isomerization of
cis,trans ML-OOH to trans,trans ML-OOH*.
Hexadecane
Hexadecane-in-
[
h]
water emulsion [h]
Control
43
96
48
0
2
0
2
.2 mM AA
120
120
43
mM AA
48
.2 mM AP
mM AP
96
73
168
*
Results are expressed as time [h] when more than 10% of the
total ML-OOH were trans,trans isomers.
Fig. 2. Effect of ascorbic acid and ascorbyl palmitate on
the decomposition of ML-OOH in hexadecane at 40 °C.
AA and AP on hydroperoxides. In our previous study we
showed that the isomerization of cis,trans ML-OOH to
trans,trans ML-OOH through peroxyl radicals precede the
decomposition of cis,trans ML-OOH [6]. In hexadecane,
Control ( ), 0.2 mM ascorbic acid (ꢀ), 2 mM ascorbic
♦
acid (ꢀ), 0.2 mM ascorbyl palmitate (ꢀ) and 2 mM ascor-
byl palmitate (ꢀ).
0.2 and 2 mM concentrations of AA inhibited the isomer-
ization of cis,trans to trans,trans ML-OOH when com-
pared to the control sample as shown in Tab. 1. Similarly,
a 2 mM level of AP either in hexadecane or in hexade-
cane-in-water emulsion slightly inhibited the isomeriza-
tion of cis,trans M-OOH to trans,trans ML-OOH (Tab. 1).
Porter et al. [23] related the ability of antioxidants (AH) to
inhibit the isomerization reaction to their ability to act as
ꢀ
hydrogen atom donors to peroxyl radicals (ROO ) as fol-
lows in (1).
ꢀ
ꢀ
ROO + AH → ROOH + A
(1)
Thus, these results suggest that, to some extent, AA and
AP act as hydrogen atom donors to peroxyl radicals and
the progress of the free radical decomposition reactions
of ML-OOH is stopped. AA and AP may also scavenge
other radicals capable of initiating the isomerization and
decomposition reactions. However, the scavenging of
peroxyl radicals is considered to be the main reaction in
inhibiting the isomerization.
Fig. 3. Effect of ascorbic acid and ascorbyl palmitate on
the decomposition of ML-OOH in hexadecane-in-water
emulsion at 40 °C. Symbols as in Fig. 2.
hexadecane-in-water emulsion (Figs. 2 and 3). Depend-
ing on the reaction conditions AA and AP either slightly in-
hibited or accelerated the decomposition of ML-OOH. In
hexadecane, AA at 0.2 and 2 mM levels inhibited the de-
composition of ML-OOH whereas AP at 2 mM accelerat-
ed the decomposition (Fig. 2). The opposite effect was
obtained in a hexadecane-in-water emulsion since AP at
Methyl linoleate hydroxy compounds are decomposition
products of hydroperoxides which do not continue the
free radical chain reactions. AP increased the formation of
methyl linoleate hydroxy compounds both in hexadecane
and in hexadecane-in-water emulsion (Tab. 2). The hy-
droxy compounds formed were cis,trans isomers, in both
models after 45 h incubation more than 97% of hydroxy
compounds were cis,trans isomers. In contrast to AP, AA
had no effect on the formation of hydroxy compounds.
These results suggest that AP reduced cis,trans ML-OOH
directly to cis,trans hydroxy compounds without a radical-
intermediated isomerization. The higher reduction capac-
ity of AP when compared to AA is possible due to the
differences in their solubility. Previously Schieberle and
0.2 and 2 mM levels inhibited the decomposition of ML-
OOH whereas AA accelerated the decomposition (Fig. 3).
These results are in accordance with the so-called ’polar
paradox’ established by Porter [16]. Other parameters be-
ing identical, nonpolar antioxidants function relatively best
in a polar lipid emulsion while polar antioxidants are rela-
tively more effective in bulk lipids. It should be noted that
all the effects of AA and AP on ML-OOH decomposition
were relatively small.
The decomposition reactions of ML-OOH were studied in
more detail to gain insight on the mechanisms of action of

686
Mäkinen et al.
Eur. J. Lipid Sci. Technol. 103 (2001) 683–687
Tab. 2. Effects of ascorbic acid (AA) and ascorbyl palmi-
tate (AP) on the amount of hydroxy compounds after 45 h
incubation of ML-OOH*.
be due to interactions with these metal ions. AA and AP
reduce transition metals to a lower valency state which
turns them into more active partners in the homolytic de-
composition of hydroperoxides compared to metals in
higher valency states [24]. The reduced form of metals
catalyzes the decomposition of hydroperoxides to reac-
tive alkoxyl radicals which may initiate new chain reac-
tions (2):
Hexadecane
Hexadecane-in-
water emulsion
Control
0.5
0.5
0.5
0.8
1.8
0.8
0.6
0.4
1.5
6.3
0
2
0
2
.2 mM AA
mM AA
n
ꢀ
–
n+1
ROOH + M → RO + HO + M
(2)
.2 mM AP
mM AP
The relevance of this reaction in vivo was recently as-
sessed by Carr and Frei [25]. They concluded that espe-
cially during tissue injury metal ions may be released from
their stores and subsequently interact with AA and hy-
droperoxides.
*
Results are expressed as % of initial ML-OOH.
Grosch [17] showed that AP inhibited the decomposition
of 13-cis,trans ML-OOH in a system containing copper(II)
ions by reducing approximately 10% of the hydroperox-
ides to their corresponding hydroxy compounds. Howev-
er, under these experimental conditions the formation of
hydroxy compounds did not have any marked effect on
the stability of ML-OOH.
4
Conclusion
In conclusion, results presented in this work showed that
depending on the reaction conditions, AA and AP were ei-
ther inhibitors or accelerators of hydroperoxide decompo-
sition. AA and AP were shown to act as hydrogen atom
donors to peroxyl radicals, reducers of hydroperoxides to
hydroxy compounds as well as accelerators of hydroper-
oxide decomposition due to their interactions with metal
ions. However, since each of these effects of AA and AP
on lipid hydroperoxides were relatively small, it is as-
sumed that the antioxidative activity of AA and AP as well
as their effect on the stability and reactions of lipid hy-
droperoxides in biological systems and in foods is mainly
due to their synergistic interactions with other antioxida-
tive compounds such as tocopherols.
The effect of metal ions on the activity of AA and AP was
studied by adding EDTA as a metal chelator in hexade-
cane-in-water emulsion. EDTA at a 50 µM level inhibited
the decomposition of ML-OOH when compared to the
control sample (Fig. 4). Also in samples containing 2 mM
of AA or AP in combination with EDTA the decomposition
rate was lower when compared to samples not containing
EDTA. This result suggests that in our model system a
small amount of metal ions catalyzed the decomposition
of ML-OOH. The slight increase in the decomposition rate
of ML-OOH in the presence of AP in hexadecane and in
the presence of AA in hexadecane-in-water emulsion may
Acknowledgements
This work was financially supported by the Research
Council for Environment and Natural Resources of the
Academy of Finland.
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