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ascorbate ratio in plasma from iron overloaded rats as oxidative stress indicator
Toxicology Letters 133 (2002) 193– 201 www.elsevier.com/locate/toxlet
Ascorbyl radical/ascorbate ratio in plasma from iron overloaded rats as oxidative stress indicator Monica Galleano *, Lucila Aimo, Susana Puntarulo Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, Uni6ersity of Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina Received 4 March 2002; received in revised form 23 April 2002; accepted 24 April 2002
Abstract Oxidative stress has been developed using dietary carbonyl-iron and iron-dextran parenteral administration as models of in vivo iron overload in rats. Carbonyl-iron led to a 2-fold increase in plasma iron content, a significant decrease (34%) in ascorbate plasma content and non-significant changes in plasma ascorbyl radical content. Iron-dextran produced a dramatic increase (6.7-fold) in plasma iron content, overwhelming the plasma total iron binding capacity. The ascorbyl radical content increased significantly in iron-dextran treatment (2.6-fold) and plasma ascorbate level was not affected. Ascorbyl radical/ascorbate ratio was significantly higher in both iron treated groups as compared with the control group (4 × 10 − 4 91×10 − 4). Data reported here indicate that the ascorbyl radical/ ascorbate ratio is an appropriate in vivo indicator of oxidative stress under conditions of iron overload. The overall mechanism that describes the ascorbate status in plasma seems to be strongly dependent on the way the excess of iron is stored and thus, to the availability of the catalytically active iron for interacting with the plasma components. On this regard, evaluation of A/AH− ratio did not help to discriminate between the possible involved mechanisms. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascorbyl radical; Ascorbate; Iron; Plasma; Oxidative stress
1. Introduction Non-enzymatic antioxidants are the primary protectants against oxidative damage in the extracellular compartment (Hubel et al., 1997). Frei et al. (1988) reported that in plasma exposed in vitro to water-soluble radical generators, such as 2,2%azobis-(2-amidino propane) dihydrochloride * Corresponding author. Tel.: + 54-11-4964-8245; fax: + 54-11-4508-3646 E-mail address: [email protected] (M. Galleano).
(AAPH), the major low molecular weight antioxidants are typically consumed in the following order: ascorbate= protein thiols\ bilirubin\ urate\ a-tocopherol. During its antioxidant action, ascorbate (AH−) undergoes two consecutive one electron oxidations to dehydroascorbic acid (DHA) with intermediate formation of the ascorbyl radical (A) (Hubel et al., 1997). A has a relatively long lifetime compared with other free radicals (approximately 50 s) (Buettner and Jurkiewicz, 1993) and it is easily detectable by electronic spin resonance (ESR) even at room
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temperature in aqueous solution. In contrast to A, AH− and DHA are ESR silent (Hubel et al., 1997). There is an increasing interest in the use of A content in biological tissues as an informative, non-invasive and natural indicator of oxidative stress (Roginsky and Stegmann, 1994). Vertebrates phylogenetically higher than fish possess gulonolactone oxidase to catalyze the final step of ascorbate generation pathway (Ba´ nhegyi et al., 1997). However, several species (man and primates in general, guinea pig, few bats and passeriform bird) do not express this enzyme, while in others species such as rat (Kawai et al., 1992) the lack of this activity is a disorder affecting only a minority of individuals. In humans, evaluation of A steady state concentrations in plasma is under active investigation and increased content has been reported associated with several oxidative stress conditions (Gey et al., 1987; Minakata et al., 1993; Pietri et al., 1994; Nakagawa et al., 1997; Courderot-Masuyer et al., 2000). However, data on A steady state concentrations in plasma of AH− synthesizing species are limited in number and interpretation (Torbati et al., 1992; Liu et al., 1994; Benderitter et al., 1998), and the effect of iron overload in vivo on A content was not characterized. It is well known that transition metals, such as iron, catalyze the oxidation of ascorbate in vitro (Buettner and Chamulitrat, 1990). In vivo iron overload is a well established model of oxidative stress in rats (Bacon and Britton, 1990). In experimental animals iron overload clearly results in hepatic lipid peroxidation (Bacon and Britton, 1990; Galleano and Puntarulo, 1992), hepatic protein oxidation (Galleano and Puntarulo, 1997; Brown and Knudsen, 1998) and oxidative damage associated to other organs including blood and/or plasma (Dabbagh et al. 1994; Galleano and Puntarulo, 1995; Brown and Knudsen, 1998). In this study oxidative stress has been developed using dietary iron-carbonyl intake and irondextran parenteral administration as experimental models of in vivo iron overload in rats. A/AH− ratio was evaluated in plasma of iron overloaded rats and compared with data obtained in control animals. Iron effect on A/AH− ratio was characterized and compared with iron-dependent modifi-
cations of other parameters used to assess oxidative damage.
2. Materials and methods
2.1. Animals and treatments Parenteral iron overload was carried out on adults male Wistar rats weighing 200–250 g that received a single i.p. injection of iron-dextran (500 mg kg − 1 body weight) (Galleano and Puntarulo, 1992). Control animals were injected with equivalent volumes of saline solution. Determinations were made 4 h after iron-dextran or saline solution administration. Dietary iron overload was carried out on male Wistar rats weighing 90–100 g that were fed during 6 weeks with either a) control chow diet (control group) or b) control chow diet supplemented with 2.5% (w/w) carbonyl-iron (iron overloaded group) (Galleano and Puntarulo, 1997). Blood was withdrawn from cardiac puncture under ether anesthesia and collected in heparinized tubes. Samples were immediately centrifuged for 5 min at 3000 g to separate plasma from blood cells.
2.2. Biochemical assays Plasma iron concentration and unsaturated iron-binding capacity (UIBC) were measured using the assay based on ferrozine (Persjin et al., 1971) purchased from Sigma Diagnostics (Procedure N° 565). Plasma ascorbate content (AH−) was measured on plasma samples that were added with metaphosphoric acid 10% (p/v) and immediately centrifuged. The supernatant was filtrated using nylon membranes (0.22 mM) and the filtrate was analyzed by electrochemical detection by reverse phase HPLC according to Kutnink et al. (1987). Thiobarbituric acid reactive substances (TBARS) content was determined fluorometrically using buthanol to extract the complex formed minimizing interferences, according to Fraga et al. (1987). a-Tocopherol (a-TH) was extracted according to Lang et al. (1986) and quantified by reverse phase HPLC with electro-
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chemical detection using a coulombimetric detector (ESA Inc., Bedford, MA, USA) at an applied oxidation potential of + 0.6 V.
2.3. ESR measurements A spectra were measured on a Bruker ECS 106 ESR, equipped with a ER 4102ST cavity, operating at the following conditions: modulation frequency 50 KHz; microwave power 20 mW; microwave frequency 9.75 GHz; centerfield at 3487 G; sweep time 84 s; time constant 655 ms per point; modulation amplitude 1G; and scan width 15 G. A concentration was determined by using 2,2,6,6-Tetramethyl piperidine-N-oxyl (TEMPO) 1 mM radical as standard. A content in control and iron overloaded rats was measured in plasma from blood samples added with 500 mM desferoxamine (DF) immediately after collection. The effect of in vitro iron supplementation on A concentration in plasma was measured according to Minetti et al. (1992). Reactions were initiated by the addition of a small volume (6 ml) from a concentrated solution of (NH4)2Fe (SO4)2.12H2O to 100 ml of control plasma (final concentration 0 –140 mM) immediately before the ESR analysis.
2.4. Statistical analysis Results shown are mean9 S.E.M. for three to six independent experiments. The data were evaluated by ANOVA and the difference between the means were assessed using the Fisher’s PLSD test.
3. Results Long term dietary iron administration (2.5% (w/w) carbonyl iron, 6 weeks) led to a 2-fold increase in plasma iron content, while short term parenteral administration as iron-dextran (500 mg kg − 1 weight, 4 h) produced a dramatic increase (6.7-fold) as compared with the iron content in plasma from control animals (Table 1). Unsaturated Iron-Binding Capacity (UIBC) is a measurement of plasma unsatured iron-binding sites, essentially provide by transferrin. Carbonyl-iron supplementation led to a 38% decrease in UIBC.
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In contrast, UIBC was drastically diminished by iron-dextran administration to less than 10% of the control value (Table 1). Subsequently, transferrin saturation percentage was increased from 33% for control group to 61% for carbonyl-iron treated group and to approximately 100% for iron-dextran treated group. Over the past decade the ESR detectable concentration of A in plasma has been interpreted either as an index of the transient changes in plasma ascorbate status (Pietri et al., 1994) or as a reflection of the ongoing free radical flux in the studied system (Jurkiewicz and Buettner, 1994; Sharma et al., 1994). In Fig. 1 is shown the typical ESR spectrum of A in plasma from control rats, with the characteristic two lines at g= 2.005 and aH + = 1.8 G. A content in plasma from control rats was not significantly increased by carbonyliron treatment but was significantly enhanced after iron-dextran administration (2.6-fold) (Table 2). It has been reported by Minetti et al. (1992) that in vitro iron supplementation (0–70 mM) to control samples from human plasma resulted in an increase in A content measured by ESR. Plasma from control rats supplemented with iron in vitro showed a pattern of response in agreement with that reported by Minetti et al. (1992) (Fig. 2). The concentration of added iron capable of producing a 50% of increase in the content of A in plasma was denominated Critical Iron Concentration (CIC) and was chosen to describe the plasma potential metal chelation ability (Gutteridge, 1989). The CIC was estimated in 68 mM for plasma from control animals. Under the experiTable 1 Iron content in plasma from control and iron overloaded rats
Control Carbonyl-iron Iron-dextran
Plasma Fe (mM)
UIBC (mM)
32 93 62 9 5a 213 928a
65 97 40 9 6a 5 92a
UIBC stands for Unsaturated Iron-Binding Capacity. In each experiment three to six animals were used. The results are indicated as mean values 9S.E.M. of three independent experiments. a Statistically different from the control group (PB0.05).
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Fig. 1. ESR spectra of ascorbyl radical. (A) Computer simulated spectra using the parameters g= 2.005 and aH + = 1.8G, and (B) spectra of control plasma samples at room temperature.
mental conditions reported here, plasma from carbonyl-iron supplemented rats exhibits lower free transferrin capacity to bound iron, and thus the estimated CIC (50 mM) was lower than that reported for control plasma but in the same range (Fig. 2). However, a completely different profile was found using plasma from iron-dextran treated animals. It was measured a drastic increase in A content in plasma upon the addition of low concentrations of iron (10 mM) and further additions did not change significantly the radical content,
Table 2 Ascorbyl radical, ascorbate and ascorbyl radical/ascorbate ratio in plasma from control and iron overloaded rats
Control Carbonyl-iron Iron-dextran
A (nM)
AH− (mM)
A/AH−×10−4
17 94 23 95 45 95a
44 9 5 29 93a 4495
49 1 89 2a 1191a
In each experiment three to six animals were used. The results are indicated as mean values 9 S.E.M. of three independent experiments. a Statistically different from the control group (PB0.05).
becoming the evaluation of CIC, as was previously defined, inapplicable to the new situation (Fig. 2). Based on (i) the lack of correlation between the oxidative damage reported after chronic iron overload and the non-significant increase of the plasmatic content of A, (ii) the remarkable effect of the in vitro iron supplementation on A content in plasma from rats overloaded with iron-dextran in vivo, and (iii) the active metabolism of AH− in rats, it was considered the need for improving the selection of an index to assess oxidative stress in plasma under these experimental conditions. The content of AH− was measured and the A/AH− ratio under the in vivo iron overload conditions was studied. Carbonyl-iron treatment led to a significant decrease (34%) in plasma AH− content, in agreement with previous reports by Dabbagh et al. (1994). Iron-dextran administration did not affect AH− content in plasma. The A/ AH− ratio in plasma was significantly higher in plasma from carbonyl-iron and iron-dextran treated rats as compared with plasma from control rats (Table 2), suggesting the development of oxidative stress conditions after both treatments. Ascorbate has a central metabolic role since it can act as an antioxidant and a prooxidant (Sadrzadeh and Eaton, 1988). Its prooxidant activity is a result of its ability to reduce metals (specially iron) to forms that react with oxygen to form initiators of lipid peroxidation (Wills, 1966), and its antioxidant activity derives from its ability to reduce peroxyl radicals that propagate lipid peroxidation or to reduce the oxidized form (a-tocopheroxyl radical) of the naturally occurring antioxidant a-tocopherol (Doba et al., 1985). According to this complex scenario, the lipid peroxidation was assessed as TBARS in plasma from control, carbonyl-iron and iron-dextran treated rats. No significant differences were measured (Fig. 3) in the iron overloaded plasma as compared with plasma from control rats. However, a-tocopherol (a-TH) content decreased by 32 and 40% in plasma from rats treated with carbonyliron and iron-dextran, respectively (Fig. 3). Assuming that TBARS content could be understood as an indicator of radical-dependent damage to lipids and a-TH content as the most efficient
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Fig. 2. Effect of in vitro addition of exogenous iron on the content of ascorbyl radical detected in plasma. Plasma from control animals ( ), carbonyl-iron treated animals (), and iron-dextran treated animals () were supplemented with iron (as Fe (NH4) (SO4)2 in 0.01 N HCl to final concentration 0 –140 mM exogenous iron. Stock iron solutions were freshly prepared every day and concentration was adjusted in order to add exactly the same volume of HCl to all the samples (including the 0 mM exogenous iron).
antioxidant protection at that level, the ratio TBARS/a-TH (damage/protection) could be considered as an index of oxidative stress in the lipid phase. The ratio was significantly increased as compared with control values by 63 and 100% in plasma from carbonyl-iron and iron-dextran treated rats (Fig. 3) as compared with control plasma. A linear correlation between the A/AH− and the TBARS/a-TH ratio is shown as the inset in Fig. 3.
4. Discussion The measurement of A concentration by itself did not seem as an effective tool to assess oxidative stress under experimental conditions of iron overload. Sasaki et al. (1984) have suggested that the value of the Keq for ascorbic acid in plasma
could be an useful index of metabolic state involving ascorbic acid as radical scavenger. More recently Courderot-Masuyer et al. (2000) have recommended the use of the ratio A/ascorbate content in plasma to assess oxidative stress, avoiding the measurement of the content of DHA content in plasma. The ratio A/ascorbate content was significantly increased by iron overload in both models tested here, indicating that oxidative stress was developed in both situations. However, in each case the index was affected following a substantially different pattern. In plasma from iron-dextran treated animals the ratio increased due to a significant increase (2.6-fold) in the content of A content without any effect on ascorbate concentration in plasma, suggesting that after 4 h of iron administration de novo synthesis and/or ascorbate enzymatic recycling might be operative. It could be hypothesized that under these experi-
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mental conditions the excess of catalytically active iron could increase ascorbate oxidation rate leading to an increased concentration of A in plasma and oxidative stress. This assumption is consistent with an overwhelmed plasma iron binding capacity (approx. 99 mM) after iron-dextran administration (total iron content 213 mM). It has been proposed that 50–70% of non-transferrin-bound iron is bound to other plasma proteins and moreover, in the presence of an iron excess it could be chelated by physiological compounds (mainly citrate and acetate) (Grootveld et al., 1989). Thus, after iron-dextran treatment, 34– 57 mM iron could be left as a redox-active form able to effectively catalyze ascorbate oxidation to A. In plasma from carbonyl-iron treated animals the ratio A/ascorbate increased presumable due to not only a slightly higher oxidation rate of ascorbate (since a gradual iron intake favored its ‘safe’ storage conditions) but to a significant decrease in
the content of ascorbate in plasma as well. Ba´ nhegyi et al. (1997) have suggested that situations of prolonged stress would increase ascorbate consumption and dehydroascorbate decomposition, and chronic iron overload by carbonyl-iron supplementation could be considered as such a condition. Besides, iron excess could affect metabolic activity in the liver decreasing the rate of ascorbate synthesis pathways. However, remains unclear why the rate of oxidation of ascorbate could be increased in plasma where the unsaturated iron binding capacity is not exceeded. In this regard, Berger et al. (1997) showed that in vitro iron addition to plasma drastically increased the AH− oxidation rate (t1/2 = 4.3 h in the presence of up to 40 mM supplemented iron and 34.7 h for control plasma), and postulated the existence of an ‘ascorbate oxidase-like’ activity in plasma represented by a catalytic redox system formed by copper atoms bound to ceruloplasmine and iron atoms
Fig. 3. TBARS and a-TH content in plasma from iron overloaded rats. Plasma TBARS (dark bars), and plasma a-TH (open bars) were measured in control, carbonyl-iron treated and iron-dextran treated animals. The ratio TBARS/a-TH (striped bars) was calculated from those data. Inset: Correlation between plasma TBARS/a-TH ratio and plasma Ascorbyl/ascorbate ratio. a Statistically different from the control group (PB0.05).
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complexed by albumin (Lovstad, 1995). This activity could be a contributing factor to the decreased AH− content in carbonyl-iron treated animals. To further analyze the AH− metabolism in plasma of iron overloaded rats as compared with control animals, the reactions involved in the generation and decay of A were considered. A is formed by AH− oxidation processes mediated or not by metal catalysts, such as iron and copper (Martell, 1982). In the absence of metals the autoxidation of biomolecules is a negligible process (Miller et al., 1990) and the reaction could be disregarded. Thus, A generation could be described by reaction 1. AH- +Fe3 + A + Fe2 + d[A] = k1[Fe3 + ][AH−] dt
reaction 1 (1)
The estimated value for the rate constant (k1) is 30 M − 1s − 1 (Roginsky and Stegmann, 1994) calculated for the reaction of AH− with Fe(III)EDTA. Catalytically active Fe concentration in plasma of control rats could be considered to reach values not higher than 0.1 mM (Carmine et al., 1995) meanwhile in plasma of carbonyl-iron treated rats could be estimated in 0.5 mM (Carmine et al., 1995), and in plasma of iron-dextran supplemented rats it could be as high as 35 mM (Grootveld et al., 1989). According to Eq. (1) and using the AH− concentrations indicated in Table 2, the rate of generation of A in plasma is 1.3 × 10 − 10, 4.4× 10 − 10 and 460× 10 − 10 M − 1 s − 1 for control, carbonyl iron and iron-dextran treated animals, respectively. Self-disproportionation has been postulated as the main or even the only way for A decay in biological systems (Roginsky and Stegmann, 1994) (reaction 2). 2A AH- +DHA − d[A] = 2 k2[A$]2 dt
reaction 2 (2)
The rate constant k2 has been estimated as 0.2−2×106 M − 1s − 1 (Sharma and Buettner, 1993; Roginsky and Stegmann, 1994). Under steady state condition for A the rate of genera-
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tion of A should be identical to the rate of decay of A, thus d[A] −d[A] = =2 k2[A$]2 dt dt
(3)
According to Eq. (3), the estimated values for k2 and the generation rate of A calculated above, the steady state concentration of A in plasma might be 8 –25; 14–46 and 151–479 nM for plasma from control, carbonyl-iron and iron-dextran treated rats, respectively. The data reported here for A steady state concentration measured by ESR are 17 94; 2395 and 459 5 nM for plasma from control, carbonyl-iron and iron-dextran treated rats, respectively. Thus, the simple kinetic model shown seems to reflect appropriately the main reactions involved in ascorbate metabolism in plasma either under physiological and chronic iron overloading conditions. However, when catalitically active iron increased drastically, such as under iron-dextran treatment, other reactions became significant and hence measured and estimated A concentration were not longer in agreement and further adjustments are required. Among the reactions that would play a role under acute increase of iron, the enzymatic recycling of ascorbate (reaction 3) (Goldenberg et al., 1993) should be pointed out 2A +NADH + H+ 2 AH− + NAD
Monodehydroascorbate reductase
reaction 3
The reaction of ascorbate with a-tocopheroxyl radical (a-T ) producing a recycling of a-tocopherol (a-TH) molecules (Buettner, 1993) is a representative process of the close association between lipid and water soluble antioxidants. This concerted action is also reflected in the linear correlation between the ratio A/AH− as oxidative stress index and the ratio TBARS/a-TH as an indicator of the balance free radical damage/antioxidant protection. Taken as a whole the data reported here indicate that the ratio A/AH− is an appropriate in vivo indicator of oxidative stress in plasma under conditions of iron overload. The overall mechanism that describes the ascorbate status in plasma
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seems to be strongly dependent on the way the excess of iron is stored and thus to the availability of the catalytically active iron for interacting with the plasma components. On this regard, evaluation of A/AH− ratio did not help to discriminate between the possible involved mechanisms.
Acknowledgements This study was supported by grants from the University of Buenos Aires, CONICET, TWAS, and Agencia Nacional de Investigacio´ n Cientı´fica y Tecnolo´ gica. S.P. and M.G. are career investigators from CONICET.
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Ascorbyl radical/ascorbate ratio in plasma from iron overloaded rats as oxidative stress indicator Monica Galleano *, Lucila Aimo, Susana Puntarulo Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, Uni6ersity of Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina Received 4 March 2002; received in revised form 23 April 2002; accepted 24 April 2002
Abstract Oxidative stress has been developed using dietary carbonyl-iron and iron-dextran parenteral administration as models of in vivo iron overload in rats. Carbonyl-iron led to a 2-fold increase in plasma iron content, a significant decrease (34%) in ascorbate plasma content and non-significant changes in plasma ascorbyl radical content. Iron-dextran produced a dramatic increase (6.7-fold) in plasma iron content, overwhelming the plasma total iron binding capacity. The ascorbyl radical content increased significantly in iron-dextran treatment (2.6-fold) and plasma ascorbate level was not affected. Ascorbyl radical/ascorbate ratio was significantly higher in both iron treated groups as compared with the control group (4 × 10 − 4 91×10 − 4). Data reported here indicate that the ascorbyl radical/ ascorbate ratio is an appropriate in vivo indicator of oxidative stress under conditions of iron overload. The overall mechanism that describes the ascorbate status in plasma seems to be strongly dependent on the way the excess of iron is stored and thus, to the availability of the catalytically active iron for interacting with the plasma components. On this regard, evaluation of A/AH− ratio did not help to discriminate between the possible involved mechanisms. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ascorbyl radical; Ascorbate; Iron; Plasma; Oxidative stress
1. Introduction Non-enzymatic antioxidants are the primary protectants against oxidative damage in the extracellular compartment (Hubel et al., 1997). Frei et al. (1988) reported that in plasma exposed in vitro to water-soluble radical generators, such as 2,2%azobis-(2-amidino propane) dihydrochloride * Corresponding author. Tel.: + 54-11-4964-8245; fax: + 54-11-4508-3646 E-mail address: [email protected] (M. Galleano).
(AAPH), the major low molecular weight antioxidants are typically consumed in the following order: ascorbate= protein thiols\ bilirubin\ urate\ a-tocopherol. During its antioxidant action, ascorbate (AH−) undergoes two consecutive one electron oxidations to dehydroascorbic acid (DHA) with intermediate formation of the ascorbyl radical (A) (Hubel et al., 1997). A has a relatively long lifetime compared with other free radicals (approximately 50 s) (Buettner and Jurkiewicz, 1993) and it is easily detectable by electronic spin resonance (ESR) even at room
0378-4274/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 1 9 6 - 0
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temperature in aqueous solution. In contrast to A, AH− and DHA are ESR silent (Hubel et al., 1997). There is an increasing interest in the use of A content in biological tissues as an informative, non-invasive and natural indicator of oxidative stress (Roginsky and Stegmann, 1994). Vertebrates phylogenetically higher than fish possess gulonolactone oxidase to catalyze the final step of ascorbate generation pathway (Ba´ nhegyi et al., 1997). However, several species (man and primates in general, guinea pig, few bats and passeriform bird) do not express this enzyme, while in others species such as rat (Kawai et al., 1992) the lack of this activity is a disorder affecting only a minority of individuals. In humans, evaluation of A steady state concentrations in plasma is under active investigation and increased content has been reported associated with several oxidative stress conditions (Gey et al., 1987; Minakata et al., 1993; Pietri et al., 1994; Nakagawa et al., 1997; Courderot-Masuyer et al., 2000). However, data on A steady state concentrations in plasma of AH− synthesizing species are limited in number and interpretation (Torbati et al., 1992; Liu et al., 1994; Benderitter et al., 1998), and the effect of iron overload in vivo on A content was not characterized. It is well known that transition metals, such as iron, catalyze the oxidation of ascorbate in vitro (Buettner and Chamulitrat, 1990). In vivo iron overload is a well established model of oxidative stress in rats (Bacon and Britton, 1990). In experimental animals iron overload clearly results in hepatic lipid peroxidation (Bacon and Britton, 1990; Galleano and Puntarulo, 1992), hepatic protein oxidation (Galleano and Puntarulo, 1997; Brown and Knudsen, 1998) and oxidative damage associated to other organs including blood and/or plasma (Dabbagh et al. 1994; Galleano and Puntarulo, 1995; Brown and Knudsen, 1998). In this study oxidative stress has been developed using dietary iron-carbonyl intake and irondextran parenteral administration as experimental models of in vivo iron overload in rats. A/AH− ratio was evaluated in plasma of iron overloaded rats and compared with data obtained in control animals. Iron effect on A/AH− ratio was characterized and compared with iron-dependent modifi-
cations of other parameters used to assess oxidative damage.
2. Materials and methods
2.1. Animals and treatments Parenteral iron overload was carried out on adults male Wistar rats weighing 200–250 g that received a single i.p. injection of iron-dextran (500 mg kg − 1 body weight) (Galleano and Puntarulo, 1992). Control animals were injected with equivalent volumes of saline solution. Determinations were made 4 h after iron-dextran or saline solution administration. Dietary iron overload was carried out on male Wistar rats weighing 90–100 g that were fed during 6 weeks with either a) control chow diet (control group) or b) control chow diet supplemented with 2.5% (w/w) carbonyl-iron (iron overloaded group) (Galleano and Puntarulo, 1997). Blood was withdrawn from cardiac puncture under ether anesthesia and collected in heparinized tubes. Samples were immediately centrifuged for 5 min at 3000 g to separate plasma from blood cells.
2.2. Biochemical assays Plasma iron concentration and unsaturated iron-binding capacity (UIBC) were measured using the assay based on ferrozine (Persjin et al., 1971) purchased from Sigma Diagnostics (Procedure N° 565). Plasma ascorbate content (AH−) was measured on plasma samples that were added with metaphosphoric acid 10% (p/v) and immediately centrifuged. The supernatant was filtrated using nylon membranes (0.22 mM) and the filtrate was analyzed by electrochemical detection by reverse phase HPLC according to Kutnink et al. (1987). Thiobarbituric acid reactive substances (TBARS) content was determined fluorometrically using buthanol to extract the complex formed minimizing interferences, according to Fraga et al. (1987). a-Tocopherol (a-TH) was extracted according to Lang et al. (1986) and quantified by reverse phase HPLC with electro-
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chemical detection using a coulombimetric detector (ESA Inc., Bedford, MA, USA) at an applied oxidation potential of + 0.6 V.
2.3. ESR measurements A spectra were measured on a Bruker ECS 106 ESR, equipped with a ER 4102ST cavity, operating at the following conditions: modulation frequency 50 KHz; microwave power 20 mW; microwave frequency 9.75 GHz; centerfield at 3487 G; sweep time 84 s; time constant 655 ms per point; modulation amplitude 1G; and scan width 15 G. A concentration was determined by using 2,2,6,6-Tetramethyl piperidine-N-oxyl (TEMPO) 1 mM radical as standard. A content in control and iron overloaded rats was measured in plasma from blood samples added with 500 mM desferoxamine (DF) immediately after collection. The effect of in vitro iron supplementation on A concentration in plasma was measured according to Minetti et al. (1992). Reactions were initiated by the addition of a small volume (6 ml) from a concentrated solution of (NH4)2Fe (SO4)2.12H2O to 100 ml of control plasma (final concentration 0 –140 mM) immediately before the ESR analysis.
2.4. Statistical analysis Results shown are mean9 S.E.M. for three to six independent experiments. The data were evaluated by ANOVA and the difference between the means were assessed using the Fisher’s PLSD test.
3. Results Long term dietary iron administration (2.5% (w/w) carbonyl iron, 6 weeks) led to a 2-fold increase in plasma iron content, while short term parenteral administration as iron-dextran (500 mg kg − 1 weight, 4 h) produced a dramatic increase (6.7-fold) as compared with the iron content in plasma from control animals (Table 1). Unsaturated Iron-Binding Capacity (UIBC) is a measurement of plasma unsatured iron-binding sites, essentially provide by transferrin. Carbonyl-iron supplementation led to a 38% decrease in UIBC.
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In contrast, UIBC was drastically diminished by iron-dextran administration to less than 10% of the control value (Table 1). Subsequently, transferrin saturation percentage was increased from 33% for control group to 61% for carbonyl-iron treated group and to approximately 100% for iron-dextran treated group. Over the past decade the ESR detectable concentration of A in plasma has been interpreted either as an index of the transient changes in plasma ascorbate status (Pietri et al., 1994) or as a reflection of the ongoing free radical flux in the studied system (Jurkiewicz and Buettner, 1994; Sharma et al., 1994). In Fig. 1 is shown the typical ESR spectrum of A in plasma from control rats, with the characteristic two lines at g= 2.005 and aH + = 1.8 G. A content in plasma from control rats was not significantly increased by carbonyliron treatment but was significantly enhanced after iron-dextran administration (2.6-fold) (Table 2). It has been reported by Minetti et al. (1992) that in vitro iron supplementation (0–70 mM) to control samples from human plasma resulted in an increase in A content measured by ESR. Plasma from control rats supplemented with iron in vitro showed a pattern of response in agreement with that reported by Minetti et al. (1992) (Fig. 2). The concentration of added iron capable of producing a 50% of increase in the content of A in plasma was denominated Critical Iron Concentration (CIC) and was chosen to describe the plasma potential metal chelation ability (Gutteridge, 1989). The CIC was estimated in 68 mM for plasma from control animals. Under the experiTable 1 Iron content in plasma from control and iron overloaded rats
Control Carbonyl-iron Iron-dextran
Plasma Fe (mM)
UIBC (mM)
32 93 62 9 5a 213 928a
65 97 40 9 6a 5 92a
UIBC stands for Unsaturated Iron-Binding Capacity. In each experiment three to six animals were used. The results are indicated as mean values 9S.E.M. of three independent experiments. a Statistically different from the control group (PB0.05).
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Fig. 1. ESR spectra of ascorbyl radical. (A) Computer simulated spectra using the parameters g= 2.005 and aH + = 1.8G, and (B) spectra of control plasma samples at room temperature.
mental conditions reported here, plasma from carbonyl-iron supplemented rats exhibits lower free transferrin capacity to bound iron, and thus the estimated CIC (50 mM) was lower than that reported for control plasma but in the same range (Fig. 2). However, a completely different profile was found using plasma from iron-dextran treated animals. It was measured a drastic increase in A content in plasma upon the addition of low concentrations of iron (10 mM) and further additions did not change significantly the radical content,
Table 2 Ascorbyl radical, ascorbate and ascorbyl radical/ascorbate ratio in plasma from control and iron overloaded rats
Control Carbonyl-iron Iron-dextran
A (nM)
AH− (mM)
A/AH−×10−4
17 94 23 95 45 95a
44 9 5 29 93a 4495
49 1 89 2a 1191a
In each experiment three to six animals were used. The results are indicated as mean values 9 S.E.M. of three independent experiments. a Statistically different from the control group (PB0.05).
becoming the evaluation of CIC, as was previously defined, inapplicable to the new situation (Fig. 2). Based on (i) the lack of correlation between the oxidative damage reported after chronic iron overload and the non-significant increase of the plasmatic content of A, (ii) the remarkable effect of the in vitro iron supplementation on A content in plasma from rats overloaded with iron-dextran in vivo, and (iii) the active metabolism of AH− in rats, it was considered the need for improving the selection of an index to assess oxidative stress in plasma under these experimental conditions. The content of AH− was measured and the A/AH− ratio under the in vivo iron overload conditions was studied. Carbonyl-iron treatment led to a significant decrease (34%) in plasma AH− content, in agreement with previous reports by Dabbagh et al. (1994). Iron-dextran administration did not affect AH− content in plasma. The A/ AH− ratio in plasma was significantly higher in plasma from carbonyl-iron and iron-dextran treated rats as compared with plasma from control rats (Table 2), suggesting the development of oxidative stress conditions after both treatments. Ascorbate has a central metabolic role since it can act as an antioxidant and a prooxidant (Sadrzadeh and Eaton, 1988). Its prooxidant activity is a result of its ability to reduce metals (specially iron) to forms that react with oxygen to form initiators of lipid peroxidation (Wills, 1966), and its antioxidant activity derives from its ability to reduce peroxyl radicals that propagate lipid peroxidation or to reduce the oxidized form (a-tocopheroxyl radical) of the naturally occurring antioxidant a-tocopherol (Doba et al., 1985). According to this complex scenario, the lipid peroxidation was assessed as TBARS in plasma from control, carbonyl-iron and iron-dextran treated rats. No significant differences were measured (Fig. 3) in the iron overloaded plasma as compared with plasma from control rats. However, a-tocopherol (a-TH) content decreased by 32 and 40% in plasma from rats treated with carbonyliron and iron-dextran, respectively (Fig. 3). Assuming that TBARS content could be understood as an indicator of radical-dependent damage to lipids and a-TH content as the most efficient
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Fig. 2. Effect of in vitro addition of exogenous iron on the content of ascorbyl radical detected in plasma. Plasma from control animals ( ), carbonyl-iron treated animals (), and iron-dextran treated animals () were supplemented with iron (as Fe (NH4) (SO4)2 in 0.01 N HCl to final concentration 0 –140 mM exogenous iron. Stock iron solutions were freshly prepared every day and concentration was adjusted in order to add exactly the same volume of HCl to all the samples (including the 0 mM exogenous iron).
antioxidant protection at that level, the ratio TBARS/a-TH (damage/protection) could be considered as an index of oxidative stress in the lipid phase. The ratio was significantly increased as compared with control values by 63 and 100% in plasma from carbonyl-iron and iron-dextran treated rats (Fig. 3) as compared with control plasma. A linear correlation between the A/AH− and the TBARS/a-TH ratio is shown as the inset in Fig. 3.
4. Discussion The measurement of A concentration by itself did not seem as an effective tool to assess oxidative stress under experimental conditions of iron overload. Sasaki et al. (1984) have suggested that the value of the Keq for ascorbic acid in plasma
could be an useful index of metabolic state involving ascorbic acid as radical scavenger. More recently Courderot-Masuyer et al. (2000) have recommended the use of the ratio A/ascorbate content in plasma to assess oxidative stress, avoiding the measurement of the content of DHA content in plasma. The ratio A/ascorbate content was significantly increased by iron overload in both models tested here, indicating that oxidative stress was developed in both situations. However, in each case the index was affected following a substantially different pattern. In plasma from iron-dextran treated animals the ratio increased due to a significant increase (2.6-fold) in the content of A content without any effect on ascorbate concentration in plasma, suggesting that after 4 h of iron administration de novo synthesis and/or ascorbate enzymatic recycling might be operative. It could be hypothesized that under these experi-
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mental conditions the excess of catalytically active iron could increase ascorbate oxidation rate leading to an increased concentration of A in plasma and oxidative stress. This assumption is consistent with an overwhelmed plasma iron binding capacity (approx. 99 mM) after iron-dextran administration (total iron content 213 mM). It has been proposed that 50–70% of non-transferrin-bound iron is bound to other plasma proteins and moreover, in the presence of an iron excess it could be chelated by physiological compounds (mainly citrate and acetate) (Grootveld et al., 1989). Thus, after iron-dextran treatment, 34– 57 mM iron could be left as a redox-active form able to effectively catalyze ascorbate oxidation to A. In plasma from carbonyl-iron treated animals the ratio A/ascorbate increased presumable due to not only a slightly higher oxidation rate of ascorbate (since a gradual iron intake favored its ‘safe’ storage conditions) but to a significant decrease in
the content of ascorbate in plasma as well. Ba´ nhegyi et al. (1997) have suggested that situations of prolonged stress would increase ascorbate consumption and dehydroascorbate decomposition, and chronic iron overload by carbonyl-iron supplementation could be considered as such a condition. Besides, iron excess could affect metabolic activity in the liver decreasing the rate of ascorbate synthesis pathways. However, remains unclear why the rate of oxidation of ascorbate could be increased in plasma where the unsaturated iron binding capacity is not exceeded. In this regard, Berger et al. (1997) showed that in vitro iron addition to plasma drastically increased the AH− oxidation rate (t1/2 = 4.3 h in the presence of up to 40 mM supplemented iron and 34.7 h for control plasma), and postulated the existence of an ‘ascorbate oxidase-like’ activity in plasma represented by a catalytic redox system formed by copper atoms bound to ceruloplasmine and iron atoms
Fig. 3. TBARS and a-TH content in plasma from iron overloaded rats. Plasma TBARS (dark bars), and plasma a-TH (open bars) were measured in control, carbonyl-iron treated and iron-dextran treated animals. The ratio TBARS/a-TH (striped bars) was calculated from those data. Inset: Correlation between plasma TBARS/a-TH ratio and plasma Ascorbyl/ascorbate ratio. a Statistically different from the control group (PB0.05).
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complexed by albumin (Lovstad, 1995). This activity could be a contributing factor to the decreased AH− content in carbonyl-iron treated animals. To further analyze the AH− metabolism in plasma of iron overloaded rats as compared with control animals, the reactions involved in the generation and decay of A were considered. A is formed by AH− oxidation processes mediated or not by metal catalysts, such as iron and copper (Martell, 1982). In the absence of metals the autoxidation of biomolecules is a negligible process (Miller et al., 1990) and the reaction could be disregarded. Thus, A generation could be described by reaction 1. AH- +Fe3 + A + Fe2 + d[A] = k1[Fe3 + ][AH−] dt
reaction 1 (1)
The estimated value for the rate constant (k1) is 30 M − 1s − 1 (Roginsky and Stegmann, 1994) calculated for the reaction of AH− with Fe(III)EDTA. Catalytically active Fe concentration in plasma of control rats could be considered to reach values not higher than 0.1 mM (Carmine et al., 1995) meanwhile in plasma of carbonyl-iron treated rats could be estimated in 0.5 mM (Carmine et al., 1995), and in plasma of iron-dextran supplemented rats it could be as high as 35 mM (Grootveld et al., 1989). According to Eq. (1) and using the AH− concentrations indicated in Table 2, the rate of generation of A in plasma is 1.3 × 10 − 10, 4.4× 10 − 10 and 460× 10 − 10 M − 1 s − 1 for control, carbonyl iron and iron-dextran treated animals, respectively. Self-disproportionation has been postulated as the main or even the only way for A decay in biological systems (Roginsky and Stegmann, 1994) (reaction 2). 2A AH- +DHA − d[A] = 2 k2[A$]2 dt
reaction 2 (2)
The rate constant k2 has been estimated as 0.2−2×106 M − 1s − 1 (Sharma and Buettner, 1993; Roginsky and Stegmann, 1994). Under steady state condition for A the rate of genera-
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tion of A should be identical to the rate of decay of A, thus d[A] −d[A] = =2 k2[A$]2 dt dt
(3)
According to Eq. (3), the estimated values for k2 and the generation rate of A calculated above, the steady state concentration of A in plasma might be 8 –25; 14–46 and 151–479 nM for plasma from control, carbonyl-iron and iron-dextran treated rats, respectively. The data reported here for A steady state concentration measured by ESR are 17 94; 2395 and 459 5 nM for plasma from control, carbonyl-iron and iron-dextran treated rats, respectively. Thus, the simple kinetic model shown seems to reflect appropriately the main reactions involved in ascorbate metabolism in plasma either under physiological and chronic iron overloading conditions. However, when catalitically active iron increased drastically, such as under iron-dextran treatment, other reactions became significant and hence measured and estimated A concentration were not longer in agreement and further adjustments are required. Among the reactions that would play a role under acute increase of iron, the enzymatic recycling of ascorbate (reaction 3) (Goldenberg et al., 1993) should be pointed out 2A +NADH + H+ 2 AH− + NAD
Monodehydroascorbate reductase
reaction 3
The reaction of ascorbate with a-tocopheroxyl radical (a-T ) producing a recycling of a-tocopherol (a-TH) molecules (Buettner, 1993) is a representative process of the close association between lipid and water soluble antioxidants. This concerted action is also reflected in the linear correlation between the ratio A/AH− as oxidative stress index and the ratio TBARS/a-TH as an indicator of the balance free radical damage/antioxidant protection. Taken as a whole the data reported here indicate that the ratio A/AH− is an appropriate in vivo indicator of oxidative stress in plasma under conditions of iron overload. The overall mechanism that describes the ascorbate status in plasma
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seems to be strongly dependent on the way the excess of iron is stored and thus to the availability of the catalytically active iron for interacting with the plasma components. On this regard, evaluation of A/AH− ratio did not help to discriminate between the possible involved mechanisms.
Acknowledgements This study was supported by grants from the University of Buenos Aires, CONICET, TWAS, and Agencia Nacional de Investigacio´ n Cientı´fica y Tecnolo´ gica. S.P. and M.G. are career investigators from CONICET.
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