Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo

Abstract

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.

Figure 1. Optical and EPR spectra following peroxide addition to methaemoglobins

(a) Optical spectra of metHbA_o (50 μM) before (Met) and 2 min after (Ferryl) the addition of 100 μM H₂O₂, with subsequent addition of 2 mM sulfide (Sulf). (b) Difference spectrum (ferryl minus met) for HbA_o (−) and Hb–DBBF (○). (c) Titration of ferryl formation (determined from absorbance change at 545 minus 700 nm) against peroxide concentration added, assuming maximum formation at saturating peroxide concentrations (○, Hb–DBBF; ■, HbA_o). Experimental conditions: sodium phosphate (50 mM) containing DTPA (diethylenetriaminepenta-acetic acid) (20 μM), pH 7.4, at 37 °C. (d) EPR spectra of metHbA_o (50 μM), before and 30 s after the addition of H₂O₂ (100 μM), in sodium phosphate (50 mM) containing DTPA (50 μM), pH 7.4, at 37 °C. EPR conditions: temperature, 10 K; microwave power, 3.2 mW; modulation amplitude, 4 G; receiver gain, 1×10⁴; microwave frequency, 9.47 GHz; time constant, 81.92 ms; scan rate, 23 G/s. (e) Close up of free radical region of EPR spectra following peroxide addition to metHbA_o and metHb–DBBF under experimental conditions identical with those in (d). Illustrated features relate to tyrosine (g=2.005) and tryptophan peroxyl radicals (g=2.033). EPR conditions: temperature, 10 K; microwave power, 12.69 μW; modulation amplitude, 3 G; microwave frequency, 9.47 GHz; receiver gain, 3.99×10⁴; time constant, 81.92 ms; number of scans, 15; scan rate, 2.9 G/s. Total radical concentrations (means±S.D.; n=6): HbA_o tyrosine, 1.31±0.09 μM; Hb–DBBF tyrosine, 1.88±0.14 μM; HbA_o tryptophan peroxyl, 0.26±0.02 μM; Hb–DBBF tryptophan peroxyl, 0.44±0.03 μM. (P<0.001 for the increase seen in both radicals in the case of Hb–DBBF.)

Figure 2. EPR spectra of blood and plasma following Hb–DBBF transfusion

(a) EPR spectrum of control rabbit venous blood measured at 10 K illustrating the following spectral features: 1. high-spin ferric haem iron in catalase, 2. high-spin ferric haem iron in methaemoglobin, 3. high-spin ferric non-haem iron in transferrin, 4. cupric copper in ceruloplasmin, 5. globin free radical. Experimental conditions: temperature, 10 K; microwave power, 3.18 mW; microwave frequency, 9.47 GHz; modulation frequency, 100 kHz; modulation amplitude, 5 G; number of scans, 1; gain, 2×10⁴; time constant, 81.92 ms; scan rate 23 G/s. (b) Venous blood before transfusion. (c) Venous blood after transfusion. (d) Venous plasma before transfusion. (e) Venous plasma after transfusion.

Figure 3. Reduction of metHb–DBBF to oxyHb–DBBF by ascorbate in vitro and in vivo

(a) Addition of ascorbate (1.5 mM) to metHb–DBBF (50 μM) in PBS at 37 °C. Lines illustrate spectra before addition and 30 and 60 min after addition. Arrows indicate change in absorbance with time. (b) Reduction of 100 μM metHb–DBBF by 100 μM ascorbate in the presence of various concentrations of erythrocytes. Control (100 μM metHb–DBBF alone) (■); +ascorbate alone (●); ascorbate+20% haematocrit (△); ascorbate+30% haematocrit (□); ascorbate+40% haematocrit (○). Results are means±S.D. (n=3), but note that, except at greater time points, the errors are less than 2.5% of the mean, thus error bars are generally not visible within the Figure. (c) Reduction of 100 μM metHb–DBBF by 100 μM ascorbate in the presence of various concentration of erythrocyte ghosts. Results are as in (b), except that ghosts were used rather than erythrocytes. Statistical analysis (see the Experimental section) revealed that ascorbate significantly increased metHb reduction and this was enhanced by the addition of erythrocytes (but not ghosts). There was no difference between the effect of 20%, 30% or 40% haematocrit. (d) Synergistic effects of ascorbate and erythrocytes on the reduction of 100 μM metHb–DBBF. Change in oxyHb–DBBF concentration with time: 40% haematocrit alone (▲); 100 μM ascorbate alone (●); 100 μM ascorbate+40% haematocrit (○).

Figure 4. Free radicals in blood and plasma before and after Hb–DBBF transfusion

Illustrative EPR spectra from venous blood and venous plasma in a rabbit transfused with Hb–DBBF: (a) venous blood before transfusion; (b) venous blood immediately after transfusion; (c) venous plasma before transfusion; (d) venous plasma immediately after transfusion; (e) venous plasma with spectral contribution of methaemoglobin at g=1.99 removed by subtraction of appropriate concentration of pure methaemoglobin spectrum. The g values indicated are those of ceruloplasmin (g=2.03), the globin free radical (g=2.005) and the low-field feature of methaemoglobin (g=1.99). EPR conditions: temperature, 10 K; microwave power, 3.18 mW; microwave frequency, 9.47 GHz; modulation frequency, 100 kHz; modulation amplitude, 3 G; number of scans, 2; gain, 1×10⁵; time constant, 40.96 ms; scan rate, 5.9 G/s.

Figure 5. Effects of ascorbate on haemoglobin radicals

(a) EPR spectra 30 s after addition of H₂O₂ (100 μM) to metHb–DBBF (50 μM) in PBS at 37 °C in the presence or absence of sodium ascorbate (200 μM), added immediately before the peroxide. Ascorbate spectrum illustrated at 5× magnification. (b) As in (a), but the experiment measured at room temperature. (c) EPR spectrum of rabbit plasma following addition of metHb–DBBF (50 μM) and H₂O₂ (100 μM); the sample was frozen 30 s after peroxide addition. Overlaid with spectrum from (a) of peroxide addition to metHb–DBBF in the presence of ascorbate. Plasma spectrum illustrated at 5× magnification. (d) As (c), but plasma pre-incubated for 30 min with ascorbate oxidase (15 units from Cucurbita sp.; Sigma Chemical Co.). Any contributions of the methaemoglobin g=1.99 signal to low-temperature studies were removed by subtraction of appropriate concentrations of the pure methaemoglobin spectrum. EPR conditions (a, c and d): temperature, 10 K; microwave power, 50 μW; microwave frequency, 9.47 GHz; modulation amplitude, 3 G; time constant, 81.92 ms; scan rate, 3.6 G/s. EPR conditions (b): temperature, 295 K; microwave frequency, 9.62 GHz; microwave power, 2 mW; modulation amplitude, 1 G; scan rate, 1.2 G/s; time constant, 81.92 ms.

Figure 6. Effects of ascorbate on ferryl haemoglobin

Optical spectral changes following H₂O₂ and ascorbate addition to metHb–DBBF. (a) H₂O₂ (50 μM) was added to metHb–DBBF (50 μM) in PBS at 37 °C. (i) metHb–DBBF, (ii) metHb–DBBF 2 min after H₂O₂ addition, (iii) metHb–DBBF 10 s after the addition of 1.5 mM sodium ascorbate, (iv) metHb–DBBF 50 s after ascorbate addition. (b) Change in absorbance at 545 nm following H₂O₂ addition to metHb–DBBF (50 μM) in PBS at 37 °C (dotted line). In the experiment illustrated by the solid line, 1.5 mM sodium ascorbate was added at the point of maximum ferryl formation.

Figure 7. Effects of ascorbate on peroxidative damage to haemoglobin

Reverse-phase HPLC chromatogram of metHb–DBBF (100 μM) reacted with H₂O₂ (200 μM) for 1 h at 37 °C in sodium acetate (20 mM), pH 5.0, (a) in the absence and (b) in the presence of 0.6 mM sodium ascorbate. The peak at 15 min is free haem b (visible at both 400 nm and 280 nm detection). The peaks at 22.7 and 31 min are from the β- and modified α-chains in Hb–DBBF. Normally, these are only detectable at 280 nm, but, in the absence of ascorbate, there is significant absorbance at 400 nm, indicating a covalent haem:protein attachment that cannot be disrupted at the pH of the HPLC column (pH 2). The optical spectrum of this species (not shown) is that previously ascribed to this covalent haem:protein species following peroxide addition to HbA_o (termed Hb-H). (c) Effect of varying the ascorbate concentration on the formation of Hb-H at pH 5.0 (in 20 mM sodium acetate) and pH 7.4 (in 20 mM sodium phosphate) at 37 °C.