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Acta Biochim Biophys
Sin 2006, 38: 58-62
doi:10.1111/j.1745-7270.2006.00127.x
Hemin-mediated Hemolysis in
Erythrocytes: Effects of Ascorbic Acid and Glutathione
Shu-De LI1,2, Yan-Dan SU3, Ming LI1, and Cheng-Gang ZOU1*
1 School
of Life Sciences, Yunnan University, Kunming 650091, China;
2 Department
of Biochemistry; and 3 Department of Clinical Chemistry, Kunming
Medical College, Kunming 650031, China
Received: July 22,
2005
Accepted: November
9, 2005
This work was supported
by a grant from the Science and Technology Research Program of Yunnan
University (No. 2004Q014B)
*Corresponding
author: Tel, 86-13312523960; Fax, 86-871-5033724; E-mail, [email protected]
Abstract In the present work, we investigated the effect
of ascorbic acid and glutathione on hemolysis induced by hemin in erythrocytes.
Ascorbic acid not only enhanced hemolysis, but also induced formation of
thiobarbituric acid-reactive substances in the presence of hemin. It has been
shown that glutathione inhibits hemin-induced hemolysis by mediating hemin
degradation. Erythrocytes depleted of glutathione became very sensitive to
oxidative stress induced by hemin and ascorbic acid. H2O2 was involved in
hemin-mediated hemolysis in the presence of ascorbic acid. However, a
combination of glutathione and ascorbic acid was more effective in inhibiting
hemolysis induced by hemin than glutathione alone. Extracellular and
intracellular ascorbic acid exhibited a similar effect on hemin-induced
hemolysis or inhibition of hemin-induced hemolysis by glutathione. The current
study indicates that ascorbic acid might function as an antioxidant or
prooxidant in hemin-mediated hemolysis, depending on whether glutathione is
available.
Key words erythrocytes; ascorbic acid; hemin; glutathione; H2O2
Heme (iron protoporphyrin IX) is a functional group of various
proteins, including hemoglobin, myoglobin, cytochromes, nitric oxide synthases
and cystathionine b-synthase [1,2]. Under some pathological conditions, such as b-thalassemia,
sickle cell anemia, glucose 6-phosphate dehydrogenase deficiency, hemorrhage,
and muscle injury, hemin (ferric protoporphyrin IX) might be released [3–5]. An excess of
hemin can then interact with the cell membrane, resulting in formation of
reactive oxygen species, cellular injury [6]. Hemin also functions as a
catalyst for the oxidation of low-density lipoprotein [7]. The toxic effects
of hemin on erythrocytes include inhibition of erythrocyte enzymes, and
dissociation of erythrocyte membrane skeletal proteins [8,9]. Hemin might cause
hemolysis by a colloid-osmotic mechanism, that is, hemolysis is preceded by the
loss of potassium from erythrocytes [10].Glutathione (GSH), the most abundant cellular antioxidant, can
protect erythrocytes against hemin-induced hemolysis [11]. Furthermore, it has
been reported that GSH itself could mediate hemin degradation by an
oxygen-dependent mechanism [12]. Ascorbic acid, another cellular antioxidant,
has been shown to inhibit oxidation of human low-density lipoprotein induced
by hemin and H2O2 and to suppress Ca2+-Mg2+-ATPase inactivation
in pink or white ghosts by t-butyl hydroperoxide and hemin [13,14]. However, ascorbic
acid can react with iron or iron-containing compounds to produce hydroxyl
radical or H2O2 [15–17]. In sickle ghosts exposed to H2O2, ascorbate
at low concentrations (<20 mM) inhibits thiobarbituric acid reactive substrates (TBARS)
production, whereas it increases TBARS production at high concentrations
(>50 mM) [18].It has been shown that GSH and ascorbic acid have synergistic
antioxidant effects on hemin-mediated cellular toxicity. The combination of
GSH and ascorbic acid inhibits lipid peroxidation in rat liver microsomes and
the degradation of folic acid induced by hemin and H2O2 [19,20]. Our
previous results demonstrated that ascorbic acid could enhance GSH-dependent
hemin degradation by increasing the production of H2O2 [21]. In the present work, we studied the effect of ascorbic acid and GSH
on hemolysis induced by hemin. Our results indicated that ascorbic acid
function as an antioxidant or prooxidant in hemin-mediated hemolysis.
Materials and Methods
Materials and reagents
Hemin was purchased from Biobasic Inc. (Markham, Canada).
3-amino-1,2,4-triazole (3-AT), catalase, 5,5‘-dithiobis (2-nitrobenzoic
acid), diethylmaleate, 2,4-dinitrophenylhydrazine, ascorbic acid, and
dehydroascorbic acid (DHA) were purchased from Sigma Chemical (St. Louis, USA).
GSH was purchased from Shanghai Dongfeng Biochemical (Shanghai, China) and
thiobarbituric acid (TBA) from Shanghai Hengxin Chemical Reagent Co.
(Shanghai, China). All other reagents were of analytic grade.Blood was obtained from healthy human volunteers by venipuncture
into heparinized tubes and centrifuged at 2000 g for 10 min at 4 ?C. The
plasma was removed and packed erythrocytes were washed three times with
phosphate-buffered saline (PBS; 10 mM sodium phosphate, 135 mM NaCl, pH 7.4).
The buffy coat of white cells was removed. The washed erythrocytes were
suspended in PBS in a final hematocrit of 5%.Hemin was freshly prepared at the beginning of each experiment as a
stock solution of approximately 1–2 mM in 5 mM NaOH and was kept in the dark on
ice. Hemin concentration was determined in 5 mM NaOH using an extinction
coefficient of 5.84?104 cm–1?M–1 at
385 nm.
Hemolysis assay
Erythrocyte suspension was incubated with various reagents at 37
?C. One milliliter of aliquots was removed and centrifuged for 3 min at 3000 g. The degree of hemolysis was determined by the absorbance of
hemoglobin at 540 nm in the supernatant. Absorbance at 100% hemolysis was
determined by adding 10 ml of Triton X-100 (10%, V/V) to 1 ml of the
erythrocyte suspension.
Measurement of TBARS
TBARS was assayed as described by Stocks and Dormandy [22]. Briefly,
0.4 ml of 28% (W/V) trichloroacetic acid and 0.1 M sodium arsenite
solution was added to 0.8 ml of erythrocyte suspension. Samples were mixed and
centrifuged at 3400 g for 5 min. We took 0.8 ml of supernatant from each
of these samples and combined them with 0.2 ml of 1% (W/V) TBA in
0.05 M NaOH to a final volume of 1.0 ml. Samples were boiled for 15 min in
capped microcentrifuge tubes and cooled on ice. The absorbance at 532 nm was
determined with quantification based upon an extinction coefficient of 1.56?105 cm–1?M –1.
Measurement of GSH
GSH in erythrocytes was assayed according to the colorimetric
method by evaluating the reduction of 5,5‘-dithiobis (2-nitrobenzoic
acid) [23]. Briefly, 1 ml of 5% erythrocyte suspension was added to 1 ml of
distilled water. Three milliliters of precipitating solution [1.67% (W/V)
glacial metaphosphoric acid, 0.2% (W/V) EDTA and 30% (W/V)
NaCl] was mixed with the hemolysate. The mixture was allowed to stand for 5 min
then filtered. We added 0.2 ml of filtrate to 0.8 ml of 0.3 M Na2HPO4
solution in a cuvette, then 0.1 ml of 0.04% (W/V) 5,5‘-dithiobis(2-nitrobenzoic
acid) in 1% (W/V) sodium citrate solution was added. The
absorbance was measured at 412 nm.
Catalase inactivation
Catalase inhibition in erythrocytes exposed to 3-AT in vitro
is a function of H2O2 flux within the cells [16]. In our experiments, 5% erythrocytes
were incubated with PBS containing 15 mM hemin, 0.2 mM ascorbic acid, 40 mM 3-AT and
1 mM diethylenetriaminepentaacetic acid at 37 ?C. At various time intervals, 1
ml of each sample was removed to microcentrifuge tubes. Erythrocytes in these
tubes were washed three times with PBS, then lysed in 1 ml of lysis buffer
(sodium phosphate buffer, 5 mM, pH 8.0). Ten microliters of lysate was added to
990 ml of 6 mM H2O2. Decomposition
of H2O2 was followed by the absorbance at 236 nm. Uninhibited catalase
activity was calculated from the mixture only containing erythrocytes and 3-AT.
Measurement of ascorbic acid
Ascorbic acid in erythrocytes was assayed as described by Omaye et
al. [24]. Briefly, 1 ml of erythrocyte suspension was added to 1 ml of
ice-cold 10% trichloroacetic acid, mixed well, and centrifuged at 3500 g
for 15 min. Then 0.5 ml of supernatant was mixed with 0.1 ml of
2,4-dinitrophenylhydrazine/thiourea/copper solution (3.0 g 2,4-dinitrophenyl-hydrazine,
0.4 g thiourea, 0.05 g CuSO45H2O in 100 ml 65%
H2SO4) and incubated for 3 h at 37 ?C. The mixture was added to 0.75 ml
of ice-cold 65% H2SO4, mixed well, and kept at room temperature for 30 min. The
absorbance was determined at 520 nm.
Statistics
Data represent mean+/–SD of five separate experiments. The paired t-test
was used to compare mean values of two groups (with and without ascorbic acid).
Multiple comparisons between groups were made by one-way ANOVA. P<0.05 was considered statistically significant.
Results and Discussion
Extracellular ascorbic acid
induces oxidative stress in the presence of hemin
Hemin is a potential hemolytic agent [10]. Approximately 27%
hemolysis was induced by 50 mM hemin after 60 min incubation with human erythrocytes. However,
the addition of ascorbic acid to erythrocytes caused an increase in hemolysis
induced by hemin, and 0.2 mM ascorbic acid is sufficient to achieve maximal
effect (data not shown). Fifty micromoles of hemin caused 37%, 43%, and 58% of
hemolysis in the presence of 0.05, 0.1, and 0.2 mM ascorbic acid, respectively
[Fig. 1(A)]. In the current study, ascorbic acid alone did not elicit
hemolysis (data not shown).The possibility that ascorbic acid might stimulate lipid
peroxidation of erythrocytes in the presence of hemin was assessed by measuring
the formation of TBARS. As shown in Fig. 1(B), hemin (50 mM) alone caused
minimal TBARS formation but greatly promoted TBARS formation in the presence of
ascorbic acid. The level of TBARS was increased with increasing concentration
of ascorbic acid [Fig. 1(B)]. TBARS formation was not detected in the
presence of ascorbic acid alone. It has not been clearly established whether
hemin promotes lipid peroxidation [25,26]. Schmitt et al. suggested that
this depends on the state of hemin [27]. At lower concentrations (<1 mM), hemin mainly
exists as monomers. Hemin aggregates with increased concentration [28].
Monomers are effective in promoting lipid peroxidation, whereas aggregates are
responsible for the increase in permeability and membrane damage. According to
Schmitt et al., there is a lack of correlation between the permeability
changes and lipid peroxidation induced by hemin [27]. The present results are consistent
with this view, as hemin itself causes hemolysis but is unable to induce
apparent formation of TBARS. Thus, the effects of hemin alone on hemolysis are
associated with the dissociation of erythrocyte membrane proteins, which
disturbed the two-layer phospholipid membrane [8,9].
H2O2 is involved in the oxidative
stress induced by hemin and ascorbic acid
It has been shown that ascorbic acid can react with iron or
iron-containing compounds to produce H2O2 [15–17]. We
therefore studied whether H2O2 is generated
by hemin and ascorbic acid. Catalase inhibition in the presence of
3-aminotriazole has been used to demonstrate H2O2
influx in erythrocytes [16]. Incubation of erythrocytes with 15 mM hemin and 0.2
mM ascorbic acid for 30 min resulted in 34% inactivation of catalase (Fig. 2),
indicating H2O2, generated from hemin and ascorbic acid at an extracelluar site,
reaches catalase within the cell. To further confirm whether H2O2 is
involved in the oxidative stress by hemin and ascorbic acid, the effect of
catalase on hemolysis and formation of TBARS was investigated. Although
catalase had no effect on hemolysis induced by hemin alone, it effectively
abolished the enhanced hemolysis as well as TBARS formation in the presence of
hemin and ascorbic acid (data not shown). These data indicate that H2O2 becomes
the primary source of oxidant stress for hemolysis and lipid peroxidation. The
net reaction leading to formation of H2O2 proceeds
as follows (Reaction 1):
Ascorbic acid+O2®H2O2+dehydroascorbic
acid 1
This reaction is catalyzed by hemin. Thus, H2O2
might participate in a Fenton reaction leading to the formation of hydroxyl
free radical (Reaction 2):
Heme-Fe2++H2O2®Heme-Fe3++HO? 2
In the current study, it is speculated that HO? is the primary
cause of hemolysis induced by hemin and ascorbic acid. However, more
experiments need to be done to prove this.
Effect of intracellular GSH on
oxidative stress mediated by hemin and ascorbic acid
To assess the effect of intracellular GSH on oxidative stress
induced by hemin and ascorbic acid, erythrocytes were depleted of GSH by
preincubation with 1 mM diethylmaleate for 60 min. Diethylmaleate depletes GSH
in a conjugation reaction catalyzed by GSH-S-transferase [29]. At this
concentration of diethylmaleate, the level of intracellular GSH in erythrocytes
decreased from 2.3 mM to 0.25 mM. This treatment did not cause further
oxidative stress in erythrocytes [29]. However, erythrocytes depleted of GSH
became very sensitive to oxidative stress induced by hemin and ascorbic acid.
Compared with normal erythrocytes, an increase in hemolysis and TBARS
formation induced by 50 mM hemin and 0.2 mM ascorbic acid was observed in erythrocytes
depleted of GSH (Table 1).We further evaluated whether replenishment of GSH by D-glucose
metabolism in the pentose cycle provides more protection of cells against
oxidative stress by hemin and ascorbic acid. As shown in Table 1, TBARS
formation induced by hemin and ascorbic acid was much lower in cells in the
presence of 0.5 mM D-glucose than in the absence of D-glucose.
GSH levels in the absence and presence of 0.5 mM D-glucose were 0.31 mM
and 1.5 mM, respectively, after erythrocytes were incubated with 50 mM hemin and 0.2
mM ascorbic acid for 60 min. These results indicate that intracellular GSH
plays an important role in protecting the cell against oxidative stress induced
by hemin and ascorbic acid.
GSH and ascorbic acid have
synergistic antioxidant effects on hemin-mediated hemolysis
Our previous results have demonstrated that ascorbic acid enhances
GSH-dependent hemin degradation, due to an increase in the production of H2O2 by
hemin and GSH [21]. In the current study, our data indicated that the combination
of GSH and ascorbic acid was more effective in inhibiting hemolysis induced by
hemin than GSH alone (Table 2). Thus, ascorbic acid might protect
erythrocytes against hemin-induced damage if enough GSH is available. According
to previous results, hemin might initially react with GSH and produce O2?–. H2O2 is generated by ascorbic acid and O2?– if ascorbic acid exists in the system, whereas H2O2 is
generated from dismutation of O2?– in the absence of ascorbic acid. Thus, ascorbic acid enhances GSH-mediated
inhibition of hemolysis induced by hemin through the production of H2O2. To
test this hypothesis, the effect of catalase on the inhibition of hemolysis by
GSH and ascorbic acid was investigated. Preincubation of catalase (100 U/ml)
with erythrocytes suppressed the inhibitory effect of GSH and ascorbic acid on
hemolysis induced by hemin (Table 2). These results imply that H2O2 is
also involved in hemin-mediated hemolysis inhibition by GSH and ascorbic acid.
Effect of intracellular
ascorbic acid on hemin-mediated hemolysis
Although ascorbic acid is transported across erythrocyte membranes
with a much lower efficiency, incubation of erythrocytes with DHA results in
marked accumulation of intracellular ascorbic acid [30]. We examined the
effect of intracellular ascorbic acid on hemolysis by hemin. After incubation
of erythrocytes (normal cells) with 0.5 mM DHA in the presence of 5 mM glucose
for 30 min, the level of intracellular ascorbic acid rose from 71 mM to 1.2 mM. The
level of intracellular GSH remained constant (2.1 mM). The erythrocytes loaded
by DHA (cells with high ascorbic acid) and normal cells were further used to
prepare GSH-depleted erythrocytes. The cells were incubated with 1 mM
diethylmaleate for 60 min. As demonstrated by May et al., diethylmaleate
treatment has no effect on concentration of intracellular ascorbic acid [29].
In our experiments, the level of ascorbic acid was 1.0 mM in the DHA-loaded
cells after diethylmaleate treatment. The level of GSH in the erythrocytes
treated with diethylmaleate (GSH-depleted cells with high ascorbic acid)
decreased to 0.29 mM. The level of GSH in normal cells was 0.19 mM when treated
with diethylmaleate (GSH-depleted cells). We next evaluated the effect of hemin on hemolysis and formation of
TBARS in four types of erythrocytes (normal cells, GSH-depleted cells, cells
with high ascorbic acid, GSH-depleted cells with high ascorbic acid) in the
presence of D-glucose. As shown in Table 3, 50 mM hemin caused
22%, 35%, 10%, and 54% hemolysis in these four types of erythrocytes,
respectively. In the presence of D-glucose, GSH was replenished by the
metabolism of D-glucose in the pentose cycle. Of these four types
of erythrocytes, hemolysis was lowest in cells with high ascorbic acid, and
highest in GSH-depleted cells. Hemin alone interacts with the erythrocyte
membrane, resulting in membrane damage and hemolysis. However, hemin easily
diffuses into erythrocytes through the cell membrane [12]. Thus, hemin can
react with intracellular ascorbic acid. The reaction might be enhanced with
time as more extracellular hemin and intracellular ascorbic acid would have a
chance to react as permeability enhancement proceeds. According to our previous
results, hemin reacts first with GSH in a system, where both GSH and ascorbic
acid exist [21]. In normal cells with high ascorbic acid, ascorbic acid
enhances the hemin degradation mediated by GSH, as there is enough GSH available
in the presence of D-glucose. Thus, of these four types of erythrocytes,
hemolysis is lowest in normal cells with high ascorbic acid (Table 3).
In contrast, hemin might react with ascorbic acid to generate H2O2 in
cells depleted of GSH with high ascorbic acid. This might explain why hemolysis
is highest in this type of erythrocyte in our experiments.
In the current study, the formation of TBARS was not consistent with
hemolysis in these four types of erythrocytes. Although TBARS was highest in
GSH-depleted cells, it was lowest in normal cells (Table 3). According
to the present data, lipid peroxidation per se is unlikely to contribute
to hemolysis induced by hemin.In conclusion, we showed that ascorbic acid alone elicits hemolysis
and induces lipid peroxidation in the presence of hemin. We also demonstrated
that H2O2 is more likely to serve as an oxidant to induce further hemolysis
and formation of TBARS. However, the combination of GSH and ascorbic acid is
more effective in inhibiting hemolysis by hemin. Although ascorbic acid itself
does not induce hemin degradation, it significantly stimulates the hemin
degradation mediated by GSH [21]. These results could provide a ready
explanation for the ascorbic acid enhancement of GSH-dependent inhibition of
hemolysis. The current study also demonstrated that extracellular and
intracellular ascorbic acid exhibit similar effects on hemin-induced hemolysis
or inhibition of hemin-induced hemolysis by GSH. Thus, ascorbic acid might
function as an antioxidant or prooxidant in hemin-mediated hemolysis, depending
on whether GSH is available.
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