Original Paper	Pdf file on Synergy omments
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 LI^1,2, 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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