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https://www.abbs.info ISSN 0582-9879 |
Detection
of Vitamin C-induced Singlet Oxygen Formation in Oxidized LDL Using MCLA as A
Chemiluminescence Probe
WANG
Juan￥,
XING Da*
( Institute of Laser Life
Science, South China Normal
University, Guangzhou 510631, China )
Abstract In this study, it was observed that addition of
vitamin C (vit C) to oxidized low-density lipoprotein (Ox-LDL) by cupric ions
(Cu2+) could result in the formation of singlet oxygen (1O2). In experiments, 1O2 was detected
by chemiluminescence method using a Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,
7-dihydroimidazo [1, 2-a] pyrazin-3-one
(MCLA), as a selective and
sensitive chemiluminescence probe.
Additional experimental evidence for the formation of 1O2
came from the quenching effect of sodium azide (NaN3) on vit
C-induced chemiluminescence in the reaction mixture of LDL-Cu2+-MCLA. Analysis based on the experimental
results demonstrated the plausible reaction mechanism is that vit C first
converts Cu2+ to its reduced state and vit C becomes vit C radical
itself, thereby stimulating the
formation of peroxyl and alkoxyl radicals, and bimolecular reaction of peroxyl radicals results in 1O2
production in the systems studied.
Key words vitamin C;
singlet oxygen; MCLA; low-density lipoprotein; Cu2+
The
lowest excited singlet oxygen (1O2) plays an important
role in biological systems[1―3]. The detection of near-infrared
spectrometry at 1.27 mm
can give precise evidence for the existence of 1O2. However, despite the advances in 1O2
detection using near-infrared spectrometry employing sensitive
semiconductor-based detectors, the
identification and direct observation of the highly reactive short-lived 1O2
in biological system remains extremely difficult because the quantum yield of
its infrared emission is 10-6 at best.
Uehara
et al.[3, 4] detected and quantified small amounts of 1O2
generated in a myeloperoxidase-H2O2-halide ion system and
heme-catalyzed decomposition of linoleic acid hydroperoxide using Cypridina
luciferin analogs,
2-methyl-6-phenyl-3, 7-dihydro-imidazo [1, 2-a] pyrazin-3-one
(CLA), and 2-methyl-6-(p-methoxyphenyl)-
3, 7-dihydroimidazo [1,
2-a] pyrazin-3-one (MCLA) as chemilumine-scence
probes in the presence of superoxide dismutase (SOD). We also found that MCLA was a very useful and sensitive
chemiluminescence probe for the identification of both superoxide anion (O2–)
and 1O2 generated in biological systems. In this study, the generation of 1O2
induced by addition of vitamin C (vit C) to oxidized low-density lipoprotein
(LDL) systems was detected by using MCLA.
The mechanism of reaction between MCLA with 1O2 or
O2– probably is producing a dioxetane analog which decarboxylates
and protonates to an excited carbonyl compound which deexcites to emit light at
465 nm[5].
Vit
C is a major water-soluble antioxidant in human plasma[6]. The use of vit C for antioxidant
therapy has been advocated because of its ability to scavenge reactive oxygen
species (ROS), which can damage
cellular macromolecules such as DNA and proteins. However, the potential
of vit C for prooxidant activity in the presence of transition metal ions has
also been recognized[7].
Ultraweak luminescence induced by vit C in Characeae cells had been
studied by Jaskowska and coworkers[8]. In this experiment,
using MCLA we observed chemiluminescence emission when vit C was added
to LDL preoxidized by cupric ions (Cu2+)[9]. Based on the selectivity of MCLA-mediated
chemiluminescence to O2– and 1O2
and the effects of various quenchers,
it appeared that the observed light emission resulted from 1O2
formation. The results showed that
together with Cu2+, vit
C probably could decompose lipid hydroperoxides by one-electron transfer
reaction and this activity might be linked to the generation of 1O2.
1 Materials and Methods
1.1
Materials
MCLA, purchased from Tokyo Kasei Kogyo
Co. Ltd., was dissolved in double-distilled water
and stored at -20 ℃
until needed. MCLA concentrations
were based upon e430
nm = 9.6 ×
103 (mol/L)-1・cm-1. Cu-Zn superoxide dismutase (SOD, from bovine erythrocytes) was obtained
from the Sigma Chemical Co.
L-ascorbic acid (vit C),
mannitol, sodium azide (NaN3), cupric sulphate 5-hydrate (CuSO4・5H2O), ethylenediaminetetracetic acid (EDTA)
and other chemicals were all of AR grade and made in China.
LDL
was separated from the healthy human serum by one-step ultracentrifugation in
discontinuous gradient according to the procedure described by Zhang et al[10]. The concentration of protein was
determined by the modified Lowry procedure described by Markwell et al[11]. In order to prevent oxidation of
LDL, all stock solutions were
bubbled with pure N2 before use.
1.2
Methods
Components
of the reaction mixture were prepared just before using. The standard reaction mixture contained
LDL (200 mg/L), Cu2+ (5
μmol/L), MCLA (2 μmol/L), vit C (1 mmol/L) and 10 mmol/L
phosphate buffer saline (PBS) at pH 7.2 in a total volume of 1.5 ml. The chemiluminescence reactions were
initiated by rapid injection of 20 μL
of vit C solution to the preoxidized LDL mixture contained 5 μmol/L
Cu2+ and 2 μmol/L
MCLA. As contrast, the chemiluminescence also were
detected when vit C was added to MCLA plus LDL pretreated with 0.2 mmol/L EDTA
to chelate trace transition metals but not preincubated with Cu2+, and when vit C was added to MCLA plus
Cu2+ in the absence of LDL.
For confirming the origin of MCLA-mediated
chemiluminescence, the peak light
intensity change was recorded after the addition of various quenchers of
reactive species to the LDL-Cu2+-vit C-MCLA reaction system. The quenchers used in experiments
included 1 μmol/L
SOD, 1 mmol/L NaN3 and
10 mmol/L mannitol.
In
order to make sure the relationship between the MCLA-mediated chemiluminescence
intensity and the concentration of conjugated diene (CD), lipid peroxidation of LDL was modulated
using different incubation times (0 h,
2 h, 4 h, 6 h and 8 h) at 37 ℃
and then chemiluminescence and CD experiments were performed at intervals of 2
h for a period of 8 h. Measurement
of CD was performed according to the literature[12] by recording the absorbance
at 234 nm of the incubation suspension of Cu2+ and LDL in 0.01 mol/L
PBS at pH 7.2.
All
chemiluminescence measurements were carried out at 25 ℃
by using a highly sensitive Intensified Charge-Coupled Device (ICCD, model: ICCD-576-s/1) detector (from Princeton Instrument Inc. USA), which was cooled to -40 ℃
by a controller. Samples in
polystyrene tubes were placed in a light-tight box. Through a photographic lens (Nikon 50 mm, f 1.4) the chemiluminescence intensity
values were collected and recorded in appropriate exposure time, then the results of chemiluminescence
measurement were displayed and processed with a Winview software in a computer.
2 Results
2.1
MCLA-mediated chemiluminescence in LDL-Cu2+-vit C systems
MCLA
can selectively react with both O2– and 1O2
generated in biological systems and O2– can be eliminated
by SOD at catalytic amounts. In
experiments, we observed that MCLA
showed appreciable spontaneous light emission by itself in the system which vit
C was omitted from the reaction solution.
However, the luminescence
could be inhibited partially by addition of 0.2 mmol/L EDTA and it was
comparatively stable, and its
level was taken as a background.
It
has been confirmed that Cu2+ can initiate the lipid peroxidation of
LDL[13, 14]. When Vit C
was added to a solution of MCLA plus LDL exposed to Cu2+, MCLA-mediated chemiluminescence
appear-ed, reached the maximum and
decreased rapidly (Fig.1).
Fig.1 MCLA-mediated chemiluminescence in
LDL-Cu2+-vit C system in 0.01 mol/L PBS of pH 7.2
LDL
(200 mg/L) preoxidized for 2 h with Cu2+ (5 μmol/L)
at 37 ℃. MCLA (2 μmol/L)
and vit C (1 mmol/L) were added at the point indicated by the arrow. CL, chemiluminescence.
In
contrast, no increase of
chemiluminescence intensity was observed when LDL was pretreated with EDTA
instead of preincubated with Cu2+ (Fig.2) or LDL was omitted from reaction solution (Fig.3)
prior to the addition of vit C.
Fig.2 MCLA-mediated chemiluminescence in
LDL-EDTA-vit C system in 0.01 mol/L PBS of pH 7.2
LDL (200 mg/L) pretreated with EDTA (0.2
mmol/L). MCLA (2 μmol/L)
and vit C (1 mmol/L) were added at the point indicated by the arrow. CL, chemiluminescence.
Fig.3 MCLA-mediated chemiluminescence in Cu2+-vit
C system in 0.01 mol/L PBS of pH 7.2
Cu2+ (5 μmol/L). MCLA (2 μmol/L)
and vit C (1 mmol/L) were added at the point indicated by the arrow. CL, chemiluminescence.
2.2 Effect of quenchers on vit C-indueced
chemiluminescence
The
addition of the O2– scavenger SOD and the・OH scavenger mannitol to the above
system, prior to the
reaction, didn’t cause a very
significant decrease (decreased by 22.5% and 8.2% respectively compared with
control) in peak chemiluminescence intensity. Results were shown in Fig.4. Considering the excellent
selectivity of MCLA to O2– and 1O2[4], the results suggested that the reaction
of vit C plus LDL exposed to Cu2+ mostly elicit the formation of 1O2
but not O2– or ・OH.
NaN3
can be used as a quencher of 1O2. In order to confirm the
1O2 formation further, the effect of NaN3 on the MCLA-mediated chemiluminescence
was observed. The peak
chemiluminescence intensity was inhibited markedly by 72.8% (Fig.4).
Fig.4 Effects of the O2–
scavenger SOD (1 μmol/L), the ・OH scavenger mannitol (10 mmol/L) and the
1O2 quencher NaN3 (1 mmol/L) on the vit C-induced
chemiluminescence intensity in LDL-Cu2+-MCLA system
LDL
(200 mg/L) preoxidized for 2 h with Cu2+ (5 μmol/L)
at 37 ℃. The quenchers were added before the
addition of vit C (1 mmol/L). Data
are presented as x±s
of three separate experiments. *P
< 0.05 in comparison with control;
** P < 0.01 in comparison with control. CL, chemiluminescence.
2.3 Correlation of chemiluminescence
intensity and CD concentration
The
preceding observations of MCLA-mediated chemiluminescence suggested that lipid
peroxidation of LDL by Cu2+ was a prerequisite for vit C-induced
light emission in the presence of MCLA.
To verify the suggestion further,
the changes of CD concentration and chemiluminescence intensity were
investigated in a set of experiments at the same time, where LDL lipid peroxidation to
different extents was used. Fig.5
shows the relationship among the CD concentration of samples, which contained LDL and Cu2+
and were incubated for varying time,
and the chemiluminescence peak intensity obtained after the addition of
vit C.
Fig.5 The relationship of vit C-induced
chemiluminescence intensity in LDL-Cu2+-MCLA system and the
concentration of CD
Lipid peroxidation of LDL was modulated
by using different incubation times (0 h,
2 h, 4 h, 6 h and 8 h) at 37 ℃
then chemiluminescence and CD measurements were performed at intervals of 2 h
for a period of 8 h. Other details
are described in the ″Materials
and Methods″.
In
Fig.5, from the linear fit of five
experimental points, the
correlation coefficient r could be calculated ( r ≈
0.96). Because the extent of CD
formation represents the extent of LDL oxidation[12], the high correlation coefficient
between chemiluminescence intensity and CD formation indicated that 1O2
generation is associated with the reaction of vit C and oxidized LDL.
3 Discussion
Dimole
1O2 emissions at 634 nm and 704 nm often have been used
for the identification of 1O2 in biological system, but unfortunately the red emission is
extremely inefficient in water[4]. On the other hand,
most of the 1O2 generated could be trapped by MCLA
at the rate constant of 2.9 ×
109 (mol/L)-1・s-1
to produce an excited dioxetane analog,
which then emits light at 465 nm with high efficiency[15]. The present study clearly indicated
that MCLA-mediated chemiluminescence is a very useful and sensitive method to
detect 1O2 generated in biological systems.
The
use of Cu2+ as a LDL oxidation catalyst has been justified[16]
because it is possibly involved in LDL oxidation in vivo, as suggested by the observation that
transition metal ions (mainly copper and iron) were found in atherosclerotic
lesion in free and protein-bound form[17]. Cu2+ is the most typical oxidant to study LDL
oxidation in vitro[9, 6]. Transition metal ions can stimulate lipid peroxidation and
also the decomposition of lipid hydroperoxides, thereby increasing peroxyl radical concentration. As a reductant, vit C has long been known to undergo
one-electron transfer to transition metal ions, and become oxidized to the ascorbyl radical.
One
pathway of termination of lipid peroxidation is the bimolecular interaction of
peroxyl radicals (Russell mechanism),
which gives rise to the formation of 1O2 and
excited carbonyls compounds,
respectively. The
generation of 1O2 by reaction between two peroxyl
radicals has previously been confirmed by its dimole emission
spectrum[12]. The Russell-type
termination of lipid peroxidation is elicited as long as lipid hydroperoxides
are present and a reductant, such
as vit C, keeps transition metals
in the reduced form.
The
experimental results and the relevant analysis suggest that the plausible
reaction scheme in the system studied is as following:
The
increase of MCLA-mediated light emission after the addition of vit C seems to
result from increased formation of 1O2 precursor LOO・.
LOO・
+ LOO・ →
LOH + LC=O* + 1O2 (1)
LOO・
may be formed from an interaction of LOOH with LO・
and vit C radical, respectively.
LOOH
+ vit C radical →
LOO・ + vit C (2)
LOOH
+ LO・ →
LOO・ + LOH (3)
The presence of vit C radical greatly
accelerates the direct formation of lipid peroxyl radicals via Equation 2.
Because the highly reactive LO・
radicals would easily oxidize LOOH to LOO・
via Equation 3, a pathway of
increasing LO・
formation by Cu+ coupled with vit C-mediated Cu2+ reduction is most
likely involved (Equation 4 and Equation 5).
LOOH
+ Cu+ →
LO・ + OH– + Cu2+ (4)
Vit
C + Cu2+ →
vit C radical + Cu+ + H+ (5)
The process of Equation 4 is analogous to
the classical Fenton reaction.
Such a reaction, depending
on the copper and its concentration,
most likely acted as the initiator of LDL lipid peroxidation in our
system. Vit C kept this reaction
running upon recycling of oxidized copper ions.
As
a chain-breaking antioxidant, vit
C probably also reduces LO・
to LOH.
LO・
+ vit C →
LOH + vit C radical (6)
This will interrupt the chain propagation
of lipid peroxidation. In
addition, scavenging of alkoxy
radicals with vit C (Equation 6) would stimulate LOO・
formation by Equation 2, and
reduce LOO・
formation by Equation 3. The phenomenon of MCLA-mediated chemiluminescence
decreasing rapidly after reaching the maximum probably is related with Equation
6.
In
summary, depending on the
MCLA-mediated chemiluminescence intensity and its relation with the extent of
lipid peroxidation in LDL (represented by the CD measurements), bimolecular reaction of peroxyl
radicals (Russell mechanism, see
Equation 1) is the most likely reaction scheme responsible for vit C-induced 1O2
formation in LDL-Cu2+ system.
References
1 Khan AU, Di Mascio P,
Medeiros MH, Wilson T. Spermine and spermidine protection of
plasmid DNA against single-strand breaks induced by singlet oxygen. Proc Natl Acad Sci USA, 1992, 89:
11428―11430
2 Deadwyler G, Sima PD, Fu
Y, Kanofsky JR. Singlet oxygen-mediated inactivation of
acetylcholinesterase: A comparison
of purified enzyme in solution and enzyme bound to K562 leukemia cells. Photochem Photobiol, 1997, 65: 884―894
3 Uehara K, Hori K, Nakano
M, Koga S. Highly sensitive chemiluminescence
method for determining myeloperoxidase in human polymorphonuclear
leukocytes. Anal Biochem, 1991, 199: 191―195
4 Koga S, Nakano M,
Uehara K. Mechanism for the
generation of superoxide anion and singlet oxygen during heme
compound-catalyzed linoleic acid hydroperoxide decomposition. Arch Biochem Biophys, 1991, 289: 223―229
5 Nakano M. Detection of active oxygen species in biological
systems. Cell Mol Neurobiol, 1998, 18(6):
565―579
6 Fu Q, Liu BW.
Oxidative modification reduces HDL-induced cholesterol efflux from
cultured human arterial smooth muscle cells. Acta Biochim Biophys Sin, 2000, 32(3): 248―252
7 Lee SH, Oe T, Blair IA.
Vitamin C-induced decomposition of lipid hydroperoxides to endogenous
genotoxins. Science, 2001, 292: 2083―2086
8 Jaskowska A, Borc R,
Milczarek I, Dudziak A, Spiewla E. Kinetics studies of ultraweak luminescence induced by
ascorbic acid in Characeae cells and their structures. Luminescence, 2001, 16: 51―56
9 Fu Q, Liu BW.
Hypochlorite and Cu2+ induced oxidation of high density
lipoprotein. Acta Biochim Biophys Sin, 2001, 33(3):
262―268
10 Zhang LH, Liu BW. Separation
of human serum lipoproteins by one-step ultracentrifugation. Acta Biochim Bioohys Sin, 1989, 21(3): 257―260
11 Markwell MA, Haas SM, Bieber
LL, Tolbert NE. A modification of the Lowry procedure
to simplify protein determination in membrane and lipoprotein samples. Anal Biochem, 1978, 87: 206―210
12 Puhl H, Waeg G,
Esterbauer H. Methods to
determine oxidation oflow-density lipoproteins. Methods Enzymol, 1994, 233: 425―441
13 Jiang Y, Liu BW.
LOOH, CD, TBARS and REM kinetic changes during
oxidation of high density lipoproteins induced by Cu2+ in vitro. Acta Biochim Biophys Sin, 1999, 31(3):
325―327
14 Yan LJ, Lodge JK, Traber
MG, Matsugo S, Packer L. Comparison between copper-mediated and hypochlorite-mediated
modification of human low density lipoproteins evaluated by protein carbonyl
formation. J lipid Res, 1997, 38: 992―1001
15 Mashiko S, Suzuki N, Koga
S, Nakano M, Goto T, Ashino T,
Mizumoto I et al.
Measurement of rate constants for quenching singlet oxygen with a Cypridina
luciferin analog (2-methyl-6-[p-methoxyphenyl]- 3, 7-dihydroimidazo[1,
2-a]pyrazin-3-one) and sodium azide.
J Biolumin Chemilumin,
1991, 6: 69―72
16 Albertini R, De Luca G,
Palladini G, Passi A, Melzi d’Eril GV, Abuja PM. Mechanistic aspects of the relationship between low-level
chemiluminescence and lipid peroxides in oxidation of low-density
lipoprotein. FEBS Lett, 1999, 459: 47―50
17 Smith C, Mitchinson MJ,
Aruoma OI, Halliwell
B. Stimulation of lipid
peroxidation and hydroxyl-radical generation by the contents of human
atherosclerotic lesions. Biochem
J, 1992, 286: 901―905
Received: August 14, 2001Accepted:
October 14, 2001
This work was supported by the National
Natural Science Foundation of China for Distinguished Young Scholars of China
(No. 69725009), and the Natural
Science Foundation of Guangdong Province (No. 000679)
￥She is studying for her doctoral degree
in Anhui Institute of Optics and Fine Mechanics, the Chinese Academy of Sciences.
*Corresponding author: Tel, 86-20-85210089;
Fax, 86-20-85216052; e-mail, [email protected]