Báo cáo khoa học: Protective effects of endomorphins, endogenous opioid peptides in the brain, on human low density lipoprotein oxidation pdf

Protective effects of endomorphins, endogenous opioid
peptides in the brain, on human low density lipoprotein
oxidation
Xin Lin
1
, Li-Ying Xue
1
, Rui Wang
1,2
, Qian-Yu Zhao
1
and Qiang Chen
1
1 Department of Biochemistry and Molecular Biology, School of Life Science, Lanzhou University, China
2 State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences,
China
It is now commonly recognized that the damaging con-
sequences of oxidative stress have been implicated in
a variety of very different human disorders, including
arteriosclerosis and diseases of the nervous system [1].
A large body of evidence indicates that the brain
appears to be particularly vulnerable to oxidative dam-
age because of its high oxygen consumption, abundant
lipid content, and relatively low antioxidant levels
[2,3]. Oxidative damage may play important roles in
the slowly progressive neuronal death that is character-
istic of several different neurodegenerative disorders,
such as Alzheimer’s disease and Parkinson disease
[4,5]. Reactive oxygen species (ROS) rapidly oxidize
cellular lipids, resulting in the formation of numerous
lipid peroxidation products in nerve cells. Oxidatively

modified lipids are able to react with cellular and sub-
cellular membranes, leading to neuronal cell death
[6,7].
Low density lipoprotein (LDL) is present in the
brain and is exposed to a highly oxygenated and
lipid-enriched environment, making it susceptible to
free radical-mediated lipid peroxidation that can
result in the formation of oxidized LDL (oxLDL)
[7–9]. A number of studies have proved that oxLDL
Keywords
antioxidant; endomorphins; free radical; lipid
peroxidation; low-density lipoprotein
Correspondence
R. Wang, State Key Laboratory for Oxo
Synthesis and Selective Oxidation, Lanzhou
Institute of Chemical Physics, Chinese
Academy of Sciences, Lanzhou 730000,
China
Fax: +86 931 8912561
Tel: +86 931 8912567
E-mail:
(Received 3 December 2005, accepted
23 January 2006)
doi:10.1111/j.1742-4658.2006.05150.x
Neurodegenerative disorders are associated with oxidative stress. Low den-
sity lipoprotein (LDL) exists in the brain and is especially sensitive to oxi-
dative damage. Oxidative modification of LDL has been implicated in
the pathogenesis of neurodegenerative diseases. Therefore, protecting LDL
from oxidation may be essential in the brain. The antioxidative effects of
endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid pep-

tides in the brain, on LDL oxidation has been investigated in vitro. The
peroxidation was initiated by either copper ions or a water-soluble initiator
2,2¢-azobis(2-amidinopropane hydrochloride) (AAPH). Oxidation of the
LDL lipid moiety was monitored by measuring conjugated dienes, thiobar-
bituric acid reactive substances, and the relative electrophoretic mobility.
Low density lipoprotein oxidative modifications were assessed by evaluat-
ing apoB carbonylation and fragmentation. Endomorphins markedly and
in a concentration-dependent manner inhibited Cu
2+
and AAPH induced
the oxidation of LDL, due to the free radical scavenging effects of endo-
morphins. In all assay systems, EM1 was more potent than EM2 and
l-glutathione, a major intracellular water-soluble antioxidant. We propose
that endomorphins provide protection against free radical-induced neuro-
degenerative disorders.
Abbreviations
AAPH, 2,2¢-azobis(2-amidinopropane hydrochloride); BHT, butylated hydroxytoluene; DNPH, 2,4-dinitrophenylhydrazine; EM1, endomorphin 1;
EM2, endomorphin 2; GSH,
L-glutathione; LDL, low density lipoprotein; oxLDL, oxidized LDL; REM, relative electrophoretic mobility; ROS,
reactive oxygen species; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substance.
FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS 1275
is cytotoxic to neurones, further suggesting that
oxLDL may not only be involved at the vascular
level of neurodegenerative diseases but also could be
more directly responsible for the degeneration of
neurones [10–12]. Elevated levels of ROS and trace
metals, such as copper and iron, capable of oxidizing
LDL are present in neurodegenerative conditions
[13]. Therefore, neuronal cells have to maintain an
effective antioxidation system in order to protect

themselves against ROS overload and subsequent
damage. As described in various excellent reviews,
antioxidants keep the fine-tuned balance between
the physiological production of ROS and their
detoxification [14,15].
Recently, it has been observed that the female sex
hormone estrogen can inhibit the oxidation of LDL
and can attenuate the cytotoxicity of oxLDL on neur-
onal cells [14,16,17]. It is believed that melatonin, a
hormone mainly secreted by the pineal gland, is an
effective free radical scavenger and antioxidant and
thus attenuates the effects of free radical-induced neur-
onal damage [18,19]. There are studies investigating
the antioxidant activity of melatonin in lipid model
systems [20] and also in LDL [21,22]. In addition, it
has been reported that the brain monoamines and their
metabolites can inhibit lipid peroxidation and protect
from oxidative damage in the brain [23]. Recent
studies have demonstrated that enkephalins (leu-enke-
phalin, met-enkephalin) and their derivatives (5-S-cyst-
einyldopaenkephalin, 2-S-cysteinyldopaenkephalin and
[d-Ala
2
, d-Leu
5
]enkephalin) have free radicals scaven-
ging activities and the capacity to reduce ROS-induced
lipid peroxidation [24–26].
Endomorphin 1 (EM1) and endomorphin 2
(EM2), endogenous opioid peptides, have been found

in much higher amounts in the human brain [27].
The two peptides differ in one amino acid: EM1
(Tyr-Pro-Trp-Phe-NH
2
) and EM2 (Tyr-Pro-Phe-Phe-
NH
2
). The major effect of the endomorphins is their
antinociceptive action [28,29]. Additionally, endomor-
phins can cause vasodilatation by stimulating nitric
oxide release from the endothelium [30] and bind
to l-opioid receptors to activate G-proteins, regulate
gastrointestinal transit, respiratory system and mem-
ory [28,31–33]. Endomorphins have been investigated
to modulate damage related to inflammatory diseases
of the brain [34]. More recently, we have found that
endomorphins can scavenge radical, and inhibit lipid
peroxidation, DNA and protein oxidative damage
[35]. It is worth to note that EM1 is more potent
than EM2 and l-glutathione (GSH). Therefore, it is
worthy to see if the antioxidant activity of endomor-
phins is also valid in LDL.
The present study investigates the preventive effects
of endomorphins against Cu
2+
- and a water-soluble
initiator 2,2¢-azobis(2-amidinopropane hydrochloride)
(AAPH)-induced human LDL lipid peroxidation
in vitro, the characteristics of which have been exten-
sively investigated. The effects of endomorphins were

compared to those of GSH. It is suggested that endo-
morphins and especially EM1 may provide antioxidant
defence in neurodegenerative disorders.
Results
Inhibition of conjugated diene formation
by EM1 and EM2
A set of representative kinetic curves of conjugated
dienes formation during the peroxidation of LDL is
shown in Fig. 1. It is seen from Fig. 1 that in the
absence of exogenous antioxidants, conjugated diene
formation was still inhibited for about 20 min (Fig. 1,
line a), demonstrating the presence of endogenous anti-
oxidants in LDL ) for example, a-tocopherol, carote-
noids, ubiquinol-10 [36] ) which can trap the initiating
and ⁄ or propagating radicals to inhibit peroxidation.
After the inhibition period, absorbance at 234 nm
increased fast with time upon Cu
2+
-initiation, indica-
ting depletion of the endogenous antioxidants and fast
peroxidation of LDL. The change in absorbance was
inhibited by addition of EM1 (1.25–5 lm), EM2
(5 lm) and GSH (5 lm) in the inhibition period. After
Fig. 1. Formation of conjugated dienes during the peroxidation of
LDL at pH 7.4 and 37 °C, initiated with Cu
2+
and inhibited by endo-
morphins and GSH. [LDL] ¼ 0.2 mg proteinÆmL
)1
;[Cu

2+
] ¼ 10 lM.
a, uninhibited peroxidation; b, inhibited with 1.25 l
M EM1; c, inhib-
ited with 2.5 l
M EM1; d, inhibited with 5 lM EM1; e, inhibited with
5 l
M EM2; f, inhibited with 5 lM GSH. The results are representa-
tive of three independent experiments.
Protective effects of endomorphins on huLDL oxidation X. Lin et al.
1276 FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS
the inhibition period the rate of the absorbance
increase became faster, which is close to the original
rate of propagation, demonstrating the exhaustion of
the antioxidant. The kinetic data deduced from Fig. 1
are listed in Table 1. It is shown in Fig. 1 and Table 1
that the inhibition period t
inh
was prolonged in a dose-
dependent fashion for EM1, resulting an about 1.6-,
2.8- and 4-fold longer interval than that of the control
at 1.25, 2.5 and 5 lm drug, respectively. On the basis
of t
inh
, R
inh
and R
p
, the antioxidant activity follows
the sequence EM1 > GSH > EM2.

Inhibition of TBARS formation by EM1 and EM2
Figure 2 shows the inhibition of thiobarbituric acid
reactive substance (TBARS) formation by EM1, EM2
and GSH during the Cu
2+
-induced peroxidation of
LDL. After 60 min incubation at 37 °C, 18 nmol
TBARSÆmg
)1
LDL protein were generated in control
experiments; TBARS were instead 15.26, 11.16, 3.84
and 2.06 nmolÆmg
)1
LDL protein in the presence of
0.625, 1.25, 2.5 and 5 lm EM1. It is clearly seen that
EM1 significantly suppressed rate of TBARS forma-
tion and increased the inhibition period in a concentra-
tion- and time-dependent manner. EM2 and GSH
show similar inhibitory activity. Figure 3 indicates the
inhibition of TBARS formation by EM1, EM2 and
GSH during AAPH-induced peroxidation of LDL. It
was found that addition of EM1 inhibited TBARS for-
mation in a dose-dependent manner. EM2 and GSH
also diminished the rate of TBARS formation and
increased the inhibition period. During both Cu
2+
-
and AAPH-induced peroxidation of LDL, the activity
sequence is EM1>GSH>EM2.
Inhibition of apoB carbonyl formation

by EM1 and EM2
Oxidative damage to protein results in the formation
of protein carbonyl groups [37]. Therefore, we meas-
ured the inhibitory effects of EM1, EM2 and GSH
on either Cu
2+
- or AAPH-induced LDL oxidation
by apoB carbonyl assay. As shown in Fig. 4,
Cu
2+
-induced apoB carbonyl formation were inhibited
about 55, 41 and 35.7% at 10, 20 and 40 lm EM1,
respectively. At the same experimental concentrations,
either EM2 or GSH were also effective. During
AAPH-induced LDL oxidation, the carbonyl content
Table 1. Kinetic parameters for the Cu
2+
-induced peroxidation of
LDL and its inhibition by endomorphins and GSH. The reaction con-
ditions and the initial concentration of the substrates are the same
as described in the legends of Fig. 1 for reactions conducted in
LDL. Data are the means of three determinations.
Compounds t
inh
(min) R
p
(10
)2
min
)1

) R
inh
(10
)3
min
)1
)
None 20.6 ± 1.6 3.8 ± 0.2 2.1 ± 0.1
EM1 (1.25 l
M) 31.9 ± 1.8** 2.3 ± 0.2 1.1 ± 0.1
EM1 (2.50 l
M) 56.6 ± 4.2** 1.9 ± 0.2 1.1 ± 0.1
EM1 (5.00 l
M) 80.6 ± 4.7** 1.5 ± 0.1 0.9 ± 0.1
EM2 (5.00 l
M) 22.5 ± 1.4 2.4 ± 0.2 3.6 ± 0.2
GSH (5.00 l
M) 54.4 ± 3.8** 2.2 ± 0.2 1.0 ± 0.1
**P<0.01 compared with the control.
Fig. 2. Inhibition of TBARS formation during the Cu
2+
-induced per-
oxidation of LDL by endomorphins and GSH at 37 °C. [LDL] ¼
0.2 mg protein ⁄ mL; [Cu
2+
] ¼ 10 lM. a, uninhibited peroxidation; b,
inhibited with 0.625 l
M EM1; c, inhibited with 1.25 lM EM1; d,
inhibited with 2.5 l
M EM1; e, inhibited with 5 lM EM1; f, inhibited

with 5 l
M EM2; g, inhibited with 5 lM GSH. Values are mean ± SE
(n ¼ 3).
Fig. 3. Inhibition of TBARS formation during the AAPH-induced per-
oxidation of LDL by endomorphins and GSH at 37 °C. [LDL] ¼
0.5 mg proteinÆmL
)1
; [AAPH] ¼ 20 mM. a, uninhibited peroxidation;
b, inhibited with 10 l
M EM1; c, inhibited with 20 lM EM1; d, inhib-
ited with 40 l
M EM1; e, inhibited with 40 lM EM2; f, inhibited with
40 l
M GSH. Values are mean ± SE (n ¼ 3).
X. Lin et al. Protective effects of endomorphins on huLDL oxidation
FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS 1277
of apoB was reduced by the addition of EM1, EM2
and GSH in a concentration-dependent manner
(Fig. 5). EM1 is more potent than EM2 and GSH,
similar to that observed in the LDL oxidation prod-
ucts assay mentioned above.
Inhibition of apoB fragmentation by EM1 and
EM2
Figures 6 and 7 show gel electrophoresis of apoB trea-
ted with EM1, EM2 and GSH in the presence of
10 lm Cu
2+
or 20 mm AAPH at 37 °C for 24 h,
respectively. The spot of apoB was observed in native
LDL (Fig. 6, lane 1), but a degradation pattern of

apoB was observed when LDL was incubated with
Cu
2+
(Fig. 6, lane 2). Compared to the Cu
2+
-treated
band in lane 2, a discernible increase in the intensity of
bands was noted with the addition of 10, 20 and
40 lm EM1 (Fig. 6A, lane 3–5). Treatment of apoB
with 40 lm EM1, EM2 and GSH in the presence of
10 lm Cu
2+
decreased the extent of apoB fragmenta-
tion (Fig. 6B). As shown in Fig. 7A, when LDL was
Fig. 4. Protection of apoB carbonyl formation during the
Cu
2+
-induced peroxidation of LDL by endomorphins and GSH. LDL
(0.5 mg proteinÆmL
)1
) in NaCl ⁄ P
i
was incubated with 10 lM Cu
2+
and ⁄ or compounds at 37 °C. After incubation for 6 h, carbonyl
content was measured as described in Experimental procedures.
Values are mean ± SE (n ¼ 3). *P<0.05 compared with GSH.
Fig. 5. Protection of apoB carbonyl formation during the AAPH-
induced peroxidation of LDL by endomorphins and GSH. LDL
(0.5 mg proteinÆmL

)1
) in NaCl ⁄ P
i
was incubated with 20 mM AAPH
and ⁄ or compounds at 37 °C. After incubation for 6 h, carbonyl
content was measured as described in Experimental procedures.
Values are the mean ± SE (n ¼ 3). *P<0.05 compared with GSH.
A 12345
B
Fig. 6. SDS ⁄ PAGE of apoB fragmentation induced by Cu
2+
and
inhibited by endomorphins and GSH. LDL (1.5 mg proteinÆmL
)1
)
was incubated with 10 l
M Cu
2+
and ⁄ or compounds in NaCl ⁄ P
i
(pH 7.4) for 24 h at 37 °C. (A) apoB fragmentation was inhibited by
EM1 in the presence of 10 l
M Cu
2+
. Lane 1, native LDL; lane 2,
0 l
M; lane 3, 10 lM EM1; lane 4, 20 lM EM1; lane 5, 40 lM EM1.
(B) apoB fragmentation was inhibited by compounds in the pres-
ence of 10 l
M Cu

2+
. Lane 1, native LDL; lane 2, 0 lM; lane 3,
40 l
M EM1; lane 4, 40 lM EM2; lane 5, 40 lM GSH.
A 12345
B
Fig. 7. SDS ⁄ PAGE of apoB fragmentation induced by AAPH and
inhibited by endomorphins and GSH. LDL (1.5 mg proteinÆmL
)1
)
was incubated with 20 m
M AAPH and ⁄ or compounds in NaCl ⁄ P
i
(pH 7.4) for 24 h at 37 °C. (A) apoB fragmentation was inhibited by
EM1 in the presence of 20 m
M AAPH. Lane 1, native LDL; lane 2,
0 l
M; lane 3, 50 lM EM1; lane 4, 100 lM EM1; lane 5, 200 lM
EM1. (B) apoB fragmentation was inhibited by compounds in the
presence of 20 m
M AAPH. Lane 1, native LDL; lane 2, 0 lM; lane
3, 200 l
M EM1; lane 4, 200 lM EM2; lane 5, 200 lM GSH.
Protective effects of endomorphins on huLDL oxidation X. Lin et al.
1278 FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS
incubated with EM1 in the presence of 20 mm AAPH
at 37 °C for 24 h, a concentration-dependent increase
in the intensity of apoB bands was observed. More-
over, we investigated the protective effects of 200 lm
EM1, EM2 and GSH against 20 mm AAPH-induced

apoB fragmentation (Fig. 7B). On the basis of the
intensity of bands, the protective activity follows the
sequence of EM1 > GSH > EM2.
Inhibition of REM by EM1 and EM2
Using the same sample solutions as used for protein
carbonyl analysis, we performed relative electropho-
retic mobility (REM) studies. Figure 8A indicates the
protective effects of EM1, EM2 and GSH against
Cu
2+
-induced LDL oxidation. In the presence of 40 or
20 lm EM1, 10 lm Cu
2+
caused only a 24.5 or 49.4%
increase in REM, respectively, but in the presence of
40 lm EM2 or GSH, 10 lm Cu
2+
caused a 95.4 or
66.2% increase in REM, respectively. Thus, EM1 is at
least twice or three times as effective as GSH or EM2 in
protecting against Cu
2+
-induced apoB modification in
LDL. As shown in Fig. 8B, exposure of LDL to 20 mm
AAPH in the presence of an increasing concentration of
EM1 (50–200 lm) resulted in a dose-dependent decrease
in REM. However, EM2 and GSH were not able to
prevent AAPH-induced apoB modification in LDL.
Discussion
It is well established that oxidative stress is not simply

a by-product of degenerative processes or the end
product of nerve cell death but may directly initiate
neurodegeneration. Although oxLDL has been studied
primarily for its role in the development of atheroscler-
osis, recent studies have identified that oxidative modi-
fication of LDL is capable of eliciting cytotoxicity,
differentiation, and inflammation in neuronal cells [10–
12,38]. Suppression of oxidative modification of LDL
by antioxidants may be effective in preventing and
treating neurodegenerative diseases [8,16]. In the pre-
sent study, two different initiating assays were used.
One is copper which is one of the major potential
sources of free radical production in the brain [39].
Cu
2+
-induced LDL peroxidation is generally believed
to involve reductive activation of Cu
2+
as the first
stage. The reductive activation may be accomplished
by a net transfer of one electron to produce Cu
+
which is a strong pro-oxidant and can rapidly generate
the ultimate initiating radicals by a Fenton-type reac-
tion with peroxides or by forming an electron transfer
complex with molecular oxygen. Another is AAPH, a
water-soluble initiator, which decomposes at physiolo-
gical temperature producing alkyl radicals (R
•
) fol-

lowed by fast reaction with oxygen to give alkyl
peroxyl radicals (ROO
•
) to initiate LDL peroxidation
(LOO
•
). In the presence of an antioxidant molecule,
AH, either the initiating peroxyl radical and ⁄ or the
propagating lipid peroxyl radical can be trapped and a
new antioxidant radical, A
•
, produced. If the A
•
is a
stabilized radical (e.g. a-tocopheroxyl radical or ascor-
bate radical) which can promote the rate-limiting
hydrogen abstraction reactions and undergo fast ter-
mination reactions, the peroxidation would be inhib-
ited [40].
The formation of lipid peroxidation products is a
phenomenon common in most types of neurone
damage associated with oxidative stress [7,10]. Cu
2+
-
induced LDL peroxidation is generally monitored by
UV spectroscopy since the primary peroxidation
Fig. 8. Inhibition of the increase in REM of LDL during the
Cu
2+
-induced (A) or AAPH-initiated (B) peroxidation of LDL by endo-

morphins and GSH. LDL (0.5 mg proteinÆmL
)1
) in NaCl ⁄ P
i
was incu-
bated with 10 l
M Cu
2+
or 20 mM AAPH and ⁄ or compounds at
37 °C. After incubation for 6 h, REM of LDL was measured as
described in Experimental procedures. Values are the mean ± SE
(n ¼ 6). *P < 0.05 and **P < 0.01 compared with GSH, respectively.
X. Lin et al. Protective effects of endomorphins on huLDL oxidation
FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS 1279
products of polyunsaturated fatty acids in LDL are
hydroperoxides possessing a conjugated diene structure
which shows characteristic UV absorption at 234 nm
[41]. The rate of the chain propagation, R
p
, the inhibited
rate of propagation by antioxidants, R
inh
, and the inhi-
bition period, t
inh
, can be easily obtained from spectro-
photometric data. In the present work, we can find from
Fig. 1 and Table 1 that on the basis of t
inh
, R

inh
and
R
p
, the antioxidant activity follows the sequence
EM1 > GSH > EM2 in conjugated diene formation.
Moreover, TBARS formation is also used to detect lipid
peroxidation products in LDL oxidation. It is clearly
seen by comparison of Fig. 1 with Figs 2 and 3 that the
antioxidant activity follows the same sequence in spite
of the activity being monitored by conjugated diene
formation or by TBARS production, and in spite of
the peroxidation being initiated by Cu
2+
or by peroxyl
radicals (generated by AAPH).
Increased tissue protein carbonyls have been detected
in numerous human diseases, such as rheumatoid arth-
ritis, ischaemia–reperfusion injury to heart muscle and
Alzheimer’s disease [42]. Protein carbonyl formation is
a biomarker of protein oxidation and has some advan-
tages over lipid peroxidation products because the for-
mation of protein-bound carbonyl groups seems to be
a common phenomenon of protein oxidation, and
because of the relatively early formation and relative
stability of oxidized proteins [42]. As shown in Figs 4
and 5, EM1 is more active than EM2 and GSH. In
addition, the present study also showed that endomor-
phins inhibited not only the lipid peroxidation of LDL
but also the fragmentation of apoB of LDL and

increase in REM in a concentration-dependent manner.
The results presented in this paper provide evidence
that endomorphins ) endogenous opioid peptides in
the brain ) are very efficient in protecting LDL
against Cu
2+
- and AAPH-induced oxidative modifica-
tion. The inhibitory activity of EM1 is much more
effective than that of tested EM2 and GSH, a major
intracellular water-soluble antioxidant. It is obvious
that these micromolar in vitro concentrations are signi-
ficantly higher than endomorphins levels normally
detected in the body. Nevertheless, the following
should be taken into account. Firstly, to reach a pro-
nounced and well-detectable oxidative damage in vitro,
one has to use rather high concentrations of oxidants
in vitro and, consequently, high concentrations of
endomorphins are necessary. Secondly, recent studies
have demonstrated that endogenous opioid peptides
are released from cells during inflammation and stress,
and reach high levels at theses sites. Finally, drugs for
effective prevention of damage or treatment may reach
pharmacological levels rather than physiological
concentrations. Moreover, the general biological activ-
ities of endomorphins act through l-opioid receptors,
whereas the antioxidant activity of endomorphins is
not dependent on opioid receptors. Very probably the
neuroprotective effects of endomorphins result from a
combination of the different modes of action.
In the case of Cu

2+
-induced LDL oxida-
tion ) where lipid peroxyl radicals are generated indi-
rectly from a series of redox reactions ) EM1 had no
apparent copper-binding effect, as judged by both
spectral study and lack of quenching of the intrinsic
drug absorption by copper (data not shown). Our
results indicate that EM1 can trap the lipid peroxyl
radicals (LOO
•
) derived indirectly from copper on the
surface of LDL particles and behave well as chain
breaking antioxidant against Cu
2+
-induced LDL per-
oxidation. Furthermore, Cu
2+
-induced LDL peroxida-
tion is considered to be more relevant to the in vivo
situation than the AAPH-induced peroxidation, since
the former most likely involves a site-specific attack of
the apoB, whereas the latter produces a more-or-less
random attack of free radicals in LDL. Also, oxida-
tively modified of LDL by Cu
2+
exhibits biological
properties very similar, if not identical, to those of
cell-oxidized LDL. In addition, a growing body of
data supports a significant role for redox active metals,
Cu, as key modulators of the pathogenic pathways

that underlie neurodegenerative disorders and oxLDL-
mediated neuronal damage in vivo, would depend on
the availability of Cu [39,43].
Recently, we have reported that endomorphins can
directly scavenge galvinoxyl radicals and AAPH-
derived alkyl peroxyl radicals [35]. The difference of
EM1 and EM2 is primarily Trp and Phe at position 3.
Trp does not possess phenolic hydrogens and only has
an indole ring, similar to melatonin ) a hormone
mainly secreted by the pineal gland ) which has been
reported to be able to protect from oxidative damage
in the central nervous system [18]. However, free
amino acids such as Trp and Phe cannot react with
galvinoxyl radicals (data not shown). Therefore, it is
suggested that both the indole ring on the Trp as well
as the side chain on the indole nucleus are essential for
the antioxidant activity of EM1. The most active
hydrogen of EM1 might be H-10 on the Trp, which is
an allylic hydrogen. It is well known that allylic hydro-
gens are very active and easily abstracted by free radi-
cals. In addition, conjugation with -NH on the indole
shall further weaken the C–H-10 bond. The mechanis-
tic details are worthy of further study.
Lipid peroxidation is increased in neurophathologi-
cal conditions such as Alzheimer’s and Parkinson
disease [10]. Recent studies have reported that oxLDL
Protective effects of endomorphins on huLDL oxidation X. Lin et al.
1280 FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS
is cytotoxic to neurones and application of antioxi-
dants may attenuate neurone death. Thus, inhibition

of LDL peroxidation by antioxidants becomes an
attractive therapeutic strategy to prevent and treat
neurodegenerative diseases. This has led to a great deal
of research devoted to the prevention of lipid peroxi-
dation in LDL by antioxidants [8,17]. However, the
great pharmacological disadvantages of many antioxi-
dants are their very limited passage through the
blood–brain barrier [14]. Therefore, the existence of
antioxidants in the brain protective systems or with
much better blood–brain barrier permeation may be
essential. The recently demonstrated powerful antioxid-
ant activities of certain amines and imines may be the
starting point for developing neuroprotective anti-
oxidants [44]. Our data demonstrate that endomor-
phins may inhibit the formation of oxLDL and thus
minimize subsequent oxLDL-induced toxicity. We pro-
pose that the neuroprotective activity of endomorphins
may provide new insights into therapeutics of neuro-
degenerative diseases and a new understanding for
oxidative stress in the brain.
Experimental procedures
Chemicals
EM1 and EM2 were synthesized in our laboratory [45]. The
purity of the compounds was determined by HPLC
(> 95%) and their structures were verified by MS and
amino acid analysis. Agarose, 2,4-dinitrophenylhydrazine
(DNPH), thiobarbituric acid (TBA) and GSH were from
Sigma (St Louis, MO). AAPH and butylated hydroxytolu-
ene (BHT) were from Aldrich (Milwaukee, WI). Sudan
Black B, Acrylamide and bis-acrylamide were from BBI

(Markham, CA). All other chemicals were of the highest
quality available.
LDL isolation
Blood collected into the anticoagulant EDTA (final concen-
tration 3 mm) was taken from healthy volunteers. LDL
(1.019–1.063 gÆmL
)1
) was isolated from the plasma by a
discontinuous density gradient centrifugation procedure as
described elsewhere [41] at 140 000 g for 6 h using a HITA-
CHI 55P-72 ultracentrifuge in KBr solution at 4 °C in the
presence of EDTA (100 lm). The isolated LDL fraction
was then dialysed with phosphate buffer (NaCl ⁄ P
i
pH 7.4)
containing 100 lm EDTA to prevent oxidation during
dialysis. EDTA was removed by dialysis with NaCl ⁄ P
i
prior
to the oxidation experiments. The concentration of protein
was determined by the method of Lowry et al. [46]. LDL
was stored in the dark at 4 °C and used within 1 week.
Determination of conjugated dienes
The ability of endomorphins to inhibit Cu
2+
-mediated
LDL oxidation was evaluated in quartz cuvettes through
continuous spectrophotometric monitoring of absorbance
increase at 234 nm, reflecting conjugated diene formation
during peroxidative processes [41]. LDL (0.2 mg pro-

teinÆmL
-1
) was incubated at 37 °C using a Shimadazu
(Kyoto, Japan) model UV-260 spectrophotometer. Oxida-
tion was initiated by 10 lm CuSO
4
. EM1, EM2 and GSH
were added to inhibit the peroxidation. The absorbance at
234 nm was measured every 5 min against appropriate ref-
erence cuvettes for the duration of the experiment. Every
experiment was repeated three times and the results were
reproducible within 10% experimental deviation.
Determination of TBARS
The formation of TBARS was used to monitor lipid per-
oxidation [47]. LDL (0.2 or 0.5 mg proteinÆmL
)1
) was
incubated at 37 °C in NaCl ⁄ P
i
, pH 7.4. The peroxidation
was initiated by either 10 lm Cu
2+
or 20 mm AAPH
and inhibited by EM1, EM2 and GSH. The reaction
mixture was gently shaken at 37 °C and aliquots of the
reaction mixture were taken out at specific intervals to
which a tricholoroacetic acid ⁄ TBA ⁄ HCl stock solution
(15% w ⁄ v trichloroacetic acid; 0.375% w ⁄ v TBA; 0.25 m
HCl) was added, together with 0.02% w ⁄ v BHT. This
amount of BHT completely prevents the formation of

any nonspecific TBARS, as well as preventing decomposi-
tion of AAPH during the subsequent boiling. The solu-
tion was heated in a boiling water bath for 15 min. After
cooling, the precipitate was removed by centrifugation.
TBARS in the supernatant was determined at 532 nm.
Results were calculated as nmol TBARSÆmg LDL pro-
tein
)1
, using a molar extinction coefficient of
156 000 m
)1
Æcm
)1
[47].
Determination of apoB carbonylation
ApoB carbonyls were measured spectrophotometrically
with the use of the carbonyl specific reagent DNPH as pre-
viously reported [48]. Briefly, LDL (0.5 mg proteinÆmL
)1
)
was incubated with either 10 lm Cu
2+
or 20 mm AAPH,
with or without different concentration of EM1, EM2 and
GSH. After incubation for 6 h at 37 °C, 0.5 mL 10 mm
DNPH in 2 N HCl was added to 1 mL of the incubation
mixture and incubated at room temperature for 1 h. Fol-
lowing addition of 0.5 mL 20% tricholoroacetic acid, the
samples were incubated on ice for 10 min and centrifuged
at 4000 g for 10 min. Protein pellets were washed three

times with 3 mL ethanol ⁄ ethyl acetate (1 : 1, v ⁄ v) and dis-
solved in 6 m guanidine (pH 2.3). The peak absorbance at
370 nm was used to quantify protein carbonyls.
X. Lin et al. Protective effects of endomorphins on huLDL oxidation
FEBS Journal 273 (2006) 1275–1284 ª 2006 The Authors Journal compilation ª 2006 FEBS 1281
Determination of apoB fragmentation
The measurement apoB fragmentation was performed by
vertical electrophoresis as described previously [49] using
a 7.5% SDS ⁄ PAGE at a constant current of 20 mA for
90 min. Oxidation of LDL (1.5 mg proteinÆmL
)1
)in
NaCl ⁄ Pi was initiated by either 10 lm Cu
2+
or 20 mm
AAPH and inhibited by EM1, EM2 and GSH. After
incubation for 24 h at 37 °C, 1 mm EDTA or 0.02%
(w ⁄ v) BHT was added to the reaction mixture to prevent
further oxidation, respectively. Then, the samples were
mixed with an equal volume of 2 · SDS ⁄ PAGE sample
buffer (100 mm Tris ⁄ HCl pH 6.8, 4% SDS, 20% glycerol,
10% b-mercaptoethanol, 0.01% Bromophenol blue),
heated at 100 °C for 5 min and loaded onto a 7.5%
acrylamide SDS-containing gel. The gels were stained
with 0.05% Coomassie Brilliant Blue R-250 and photo-
graphed.
Determination of REM
The negative surface charge of apoB was determined by
agarose gel electrophoresis as described previously [49].
LDL (0.5 mg proteinÆmL

)1
) was incubated with either
10 lm Cu
2+
or 20 mm AAPH, with or without different
concentration of EM1, EM2 and GSH. After incubation
for 6 h at 37 °C, the samples were examined by electro-
phoresis at 100 V for 30 min in 50 mm barbital buffer
(pH 8.6) on 0.5% agarose gels and stained with Sudan
Black B. The REM was defined as the ratio of migrating
distance of oxidized LDL to that of native LDL.
Statistical analysis
Results are expressed as mean ± SE. For most experi-
ments, mean values were compared using Student’s t-test
to evaluate statistical differences. In the figures, symbols *
and ** indicate P < 0.05 and P < 0.01, respectively.
Acknowledgements
We thank the National Natural Science Foundation of
China (no. 20372028), the Ministry of Science and
Technology (no. 2002CCC00600 and 2003AA2Z3540),
the Teaching and Research Award Program for Out-
standing Young Teachers, and Specialized Research
Fund for the Doctoral Program in Higher Education
Institution of the Ministry of Education of China for
financial support.
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