Bioconjugate Chem. 1997, 8, 539−544
539
Methemoglobin Formation in Hemoglobin Vesicles and Reduction by Encapsulated Thiols Shinji Takeoka, Hiromi Sakai, Takehiro Kose, Yuichi Mano, Yuriko Seino, Hiroyuki Nishide, and Eishun Tsuchida*,† Department of Polymer Chemistry, Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169, Japan. Received December 23, 1996X
The hemoglobin vesicle (HbV) is a red cell substitute encapsulating purified concentrated Hb in a phospholipid vesicle. In order to suppress metHb formation or autoxidation, for the long-term maintenance of the oxygen transporting capability, a series of thiols (cysteine, Cys; glutathione, GSH; homocysteine, Hcy; and acetylcysteine, Acy) were studied as reductants of metHb. Hcy and GSH showed a good suppressive effect on metHb formation, while Cys adversely accelerates the metHb formation at a rate twice that of the Hb solution without any reductants and Acy showed no change. The significant suppression by the coaddition of superoxide dismutase (SOD) and catalase to Cys indicated that Cys was easily oxidized by oxygen and simultaneously generates a large amount of active oxygens. The effective suppression of metHb formation by SOD and catalase was not observed for HbV containing no reductants, indicating that the generation of active oxygens from Hb itself is not significant. The coencapsulation of Hcy with Hb resulted in a low rate of metHb formation in HbV (initial rate, 1%/h) in vitro at an oxygen partial pressure (PO2) of 142 Torr. The rate increased with decreasing PO2, showed a maximum (2.2%/h) around PO2 ) 23 Torr, and then decreased to 0%/h at 0 Torr. From these results, it is suggested that the fast metHb formation rate in the blood circulation of Wistar rats injected with 20 vol % of the HbV solution would be mainly caused by the exposure of HbV to the low PO2.
INTRODUCTION
The hemoglobin vesicle (HbV)1 or liposome-encapsulated hemoglobin (LEH), which have the cellular structure of a phospholipid vesicle encapsulating a concentrated Hb solution, is expected as one candidate for red cell substitutes for transfusion (Djordjevich & Miller, 1980; Hunt et al., 1985; Rudolph, 1995; Tsuchida & Takeoka, 1995). HbV with excellent physicochemical properties was prepared (Sakai et al., 1996; Takeoka et al., 1996), and a 90% blood exchange transfusion was carried out with the HbV using rats (Izumi et al., 1997; Sakai et al., 1997). On the other hand, acellular Hb solutions such as chemically modified Hb or recombinant Hb are now under clinical trials (Winslow, 1995); however, some side effects were reported such as vasoconstriction causing transient hypertension, which were considered to be induced by the trapping of nitric oxide generated from endothelial cells by acellular Hb (Vandegriff & Winslow, 1995; Tsai et al., 1996; Olsen et al., 1996). They will be solved by encapsulating Hb with a membrane. The common issue of Hb-based red cell substitutes is the relatively rapid metHb formation during blood circulation (Yang & Olsen, 1989; Stratton et al., 1988) due to the absence of metHb reduction systems originally present in a red blood cell. The systems include NADH* To whom all correspondence should be addressed. Fax: +813-5286-3120. Phone: +813-3205-4740. † CREST investigator, JST. X Abstract published in Advance ACS Abstracts, July 1, 1997. 1 Abbreviations: Hb, hemoglobin; HbV, hemoglobin vesicles; RBC, red blood cell; metHb, methemoglobin; HbCO, carbonylhemoglobin; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol; PBS, phosphate-buffered saline; PO2, oxygen partial pressure; Cys, L-cysteine; Hcy, DL-homocysteine; GSH, glutathione; Acy, N-acetyl-L-cysteine.
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cytochrome b5, NADPH-flavin, direct reduction by glutathione (GSH) and ascorbic acid, and scavengers for active oxygens such as superoxide dismutase (SOD) for O2- and catalase for H2O2. Hb in a ferrous state is autoxidized to the ferric state (metHb) and loses its oxygen binding ability. It is well-known that HbO2 dissociates into metHb and O2- (Brunori et al., 1975; Tomoda et al., 1981; Wallace et al., 1982; Shikama, 1984; Watkins et al., 1985); however, the percentage of metHb in RBC is maintained at less than 0.5% of the total Hb by the systematic reduction. Preservation of the native enzyme activities in RBC is one method of reducing the metHb formation in HbV (Ogata et al., 1995); however, their activity would change with the conditions of outdated red blood cells and the amounts of remaining substances, and virus inactivation using heat treatment is impossible in this case. Moreover, the mechanism of metHb reduction is complicated and influenced by many unknown factors. In the case of modified Hb solutions, direct conjugation with catalase and superoxide dismutase to suppress the metHb formation was reported by Quebec and Chang (1995); however, diminution of active oxygens is not sufficient to maintain the low metHb content. Especially in our case, these chemicals and enzymes are denatured and completely removed during the purification of Hb from RBC (Sakai et al., 1993) and the preparation of HbV; the construction of a metHb reduction system by coencapsulation of an appropriate amount of reductants is required. There are many reductants which can reduce the metHb to deoxyHb under anaerobic conditions. However, a few of them can suppress the metHb formation of Hb vesicles; nevertheless, some enhance the metHb formation. It is because under aerobic conditions there is no metHb to be reduced in the Hb vesicle at the beginning, and encapsulated reductants are autoxidized generally faster than the rate of metHb formation. Furthermore, active oxygens generated from the autoxidation of the © 1997 American Chemical Society
540 Bioconjugate Chem., Vol. 8, No. 4, 1997
Figure 1. Estimated elemental reactions in the mixture of Hb and thiols.
reductant oxidize the Hb to metHb (Sampath & Caugey, 1985; Eyer et al., 1975). Therefore, a relatively high stability under aerobic condition is desired for reductants. Farivre et al. (1994) reported the coinjection of reductants such as ascorbic acid and methylene blue with modified Hb solutions; however, such small molecules are rapidly excreted from the kidney. Glutathione (GSH), a kind of thiol compound, is a representative reductant for reducing metHb nonenzymatically (12 mM in a red blood cell). We found that GSH had the effective activity to suppress the metHb formation in the Hb vesicle (Sakai et al., 1994). In this paper, the encapsulation effect of a series of thiols, including GSH, on the reduction of metHb was studied from the view point of their structures and the elemental reactions in terms of active oxygens and oxygen partial pressures. The metHb formation of HbV during blood circulation was also studied to confirm the effectiveness of the selected reductant for the suppression of metHb formation in HbV in vivo. EXPERIMENTAL PROCEDURES
Purification of Hemoglobin from Outdated Red Blood Cells. Hemoglobin was simply isolated from outdated red blood cells (Hokkaido Red Cross Blood Center) using our original processes (Sakai et al., 1993): (1) stabilization of Hb using carbonylation, (2) solvent treatment for hemolysis and removal of stromata, (3) removal of residual solvent with active carbon, (4) heat treatment (60 °C for 10 h), (5) dialysis to remove the small molecules, and (6) ultrafiltration (cutoff MW of 30 000). The profile of the obtained HbCO solution is as follows: [Hb], 40 g/dL; [metHb], Acy. The thiol group dissociates to a thiolate anion, and one electron transfers from it to metHb, resulting in the production of deoxyHb and disulfide. On the other hand, the apparent rate constants of thiol oxidation by oxygen
MetHb Reduction by Thiols in Hb Vesicles
Figure 2. (a) Representative visible spectral change of metHb after the addition of thiol (Acy, 63 mM) under anaerobic conditions [[metHb] ) 8 µM in HEPES buffer (50 mM, pH 7.0)]. (b) Kinetic analysis of metHb reduction rates [[metHb] ) 10 µM, [thiols] ) 20-60 mM in HEPES buffer (50 mM, pH 7.0)]: (O) Cys, (4) Hcy, (0) GSH, (b) Acy, and ([metHb]int) initial concentration of metHb.
(k2app) were measured using an N-ethylmaleimide method and are also listed in Table 1. Cys showed the highest value, and the order is Cys . Hcy > Acy > GSH. It is generally expected that the thiol with the higher dissociation degree leads to the higher reactivity. However, it is difficult to obtain the dissociation constant of the thiol group, because pH titration is also accompanied by the deprotonation of the ammonium group. The obtained equilibrium constant contains both the proton dissociation of the thiol group and the deprotonation of the ammonium group. The partial charge of hydrogen in an SH group of the thiols was calculated because the higher value indicates the higher tendency of the proton dissociation. First, the structures of Cys, Hcy, GSH, and Acy were optimized on the basis of molecular force field calculations using the MM2 parameter. The molecular orbital was calculated by a semiempirical method using the PM3 parameter, and then the partial charge was calculated. This calculation was performed using CAChe Mechanics (version 3.8) and MOPAC 94 (version 3.8) (Sony Tektronix Co.). Figure 3 shows the rate of metHb reduction (k1app) and the rate of thiol autoxidation (k2app) plotted versus the calculated partial charge. In this figure, the partial charge of Acy was very low (-0.005) and could not be depicted. The order of the partial charge is Cys > Hcy > GSH . Acy. Interestingly, the effect of the partial charge is more significant for thiol autoxidation than for metHb reduction. Since the order of the dissociation constant from the literature is Cys > GSH > Hcy > Acy (Friedman et al., 1965), the order of the rate constants for metHb reduction and the thiol oxidation agrees well with the order of the dissociation constants except for GSH. From the view point of molecular structure, it is supposed that the dissociation of the thiol group of Cys is promoted by the existence of the ammonium group which shields the electrostatic repulsion between the thiolate anion and carboxylate anion, while the thiol dissociation of Acy is quite low because such an amino group is acetylated. In the case of Hcy, the shielding effect of the ammonium group is lower than that of Cys. GSH shows the slower rate of metHb reduction in spite of its dissociation rate being higher than that of Hcy. The thiolate anion of GSH would be stabilized by the neighboring electrophilic carbonyl group, and reactions with both metHb and oxygen would be restricted. Figure 4 shows the time courses of metHb formation in 40 g/dL Hb solutions in the presence of thiols. The addition of Cys (10.0 mM) to the Hb solution (6.2 mM) promoted metHb formation and showed an initial rate
Bioconjugate Chem., Vol. 8, No. 4, 1997 541
of metHb formation 6 times higher in comparison with that of the Hb solution without Cys. Acy does not show the ability to reduce metHb. Both GSH and Hcy showed the effective suppression of metHb formation to 70 and 60%, respectively, of the initial rate of metHb formation for the Hb solution without reductants (Table 1). Considering the balance of the Cys contribution toward metHb reduction and autoxidation by oxygen, it is postulated from Figure 4 that the generation of active oxygens such as O2- and H2O2 during the autoxidation of Cys would adversely convert Hb to metHb because the autoxidation rate is high (Misra, 1974). This has been confirmed by the addition of superoxide disumutase (SOD) and catalase with Cys, after which the rate of metHb formation dramatically decreased from 7.5 to 0.9 M-1 min-1 (Table 1). This addition effect is due to the quenching of the active oxygens such as O2- and H2O2 generated from Cys. On the other hand, Hcy and GSH showed the effective suppression of metHb formation because their low rates of autoxidation exceed the low rate of metHb reduction. The fact that the rate of metHb formation slightly decreased from 0.7 to 0.6 M-1 min-1 even in the presence of SOD and catalase was supported. GSH showed a suppressive effect of metHb formation lower than that of Hcy; however, the effect was maintained for a longer time than that of Hcy because GSH is less reactive. The above-mentioned results suggest that it is important to consider both the metHb reduction and the autoxidation of the reductants. Because the metHb concentration to be reduced by reductants is small at the initial stage, most of the reductants should be autoxidized accordingly. A suitable reductant should possess a small rate of autoxidation but a high efficiency of metHb reduction. This can be expressed from k2app/ k1app, and its order is Acy > Cys > GSH > Hcy (Table 1). From the above results, we selected Hcy as a reductant in HbV to effectively suppress metHb formation. In order to determine the appropriate concentration of Hcy in the inner Hb solution of HbV, the rate of metHb formation was studied for Hb solutions with various Hcy concentrations. The results are shown in Figure 5. The rate of metHb formation decreases with increasing Hcy concentration and then shows a minimum at around 5-10 mM. In the high Hcy concentration region, the active oxygens generated by the autoxidation of Hcy should accelerate metHb formation, and this effect exceeds the effect of metHb reduction. Therefore, an Hb solution containing 5 mM Hcy was used for encapsulation. However, in the presence of SOD and catalase, the decrease in the metHb percentage was observed for the higher concentration of Hcy because the active oxygens from the autoxidation of Hcy would be diminished by SOD and catalase. In this case, over 10 mM Hcy is effective, and the rate of metHb formation was 0.1%/h at 37 °C. There is no significant difference between the SOD- and catalase-coadded system and the catalaseadded one, indicating that Hcy does not generate O2- but H2O2. Effect of Hcy and Catalase on the MetHb Formation in HbV in Vitro. The metHb formation in HbV containing 5 mM Hcy was studied in vitro at a PO2 of 143 Torr and 37 °C, and the metHb content was 23% after 24 h (Figure 6, ]). This rate was 60% of the rate in the case of HbV without Hcy (35% after 24 h). As for the modified Hb solutions, chemical modification sometimes decreases the stabilization of Hb against oxidation; e.g., polymerized Hb shows 80% metHb after 24 h (Quebec & Chang, 1995). The advantage of HbV is coencapsulation
542 Bioconjugate Chem., Vol. 8, No. 4, 1997
Takeoka et al.
Table 1. Dissociation Constants (pKa) and Rate Constants of Elemental Reactions of Thiols and Their Influence on the Rate of MetHb Formationa thiol L-cysteine
(Cys) glutathione (GSH) homocysteine (Hcy) N-acetylcysteine (Acy) control a
b
pKab,c
metHb reduction k1app (M-1 min-1)
thiol oxidation k2app (M-1 min-1)
k2app/k1app
d[metHb] dtt)0 (M min-1)
d[metHb] dtt)0 (M min-1)
8.15 8.56 8.70 9.52 -
1.0 0.15 0.78 0.11 -
5.4 0.37 1.7 0.73 -
5.4 2.5 2.2 6.6 -
7.5 0.9 0.7 1.3 1.2
0.9 0.6 -
[Hb] ) 6.2 mM. [thiol] ) 10 mM. [PLP] ) 18.6 mM in HEPES buffer (50 mM, pH 7.0). PO2 ) 142 Torr at 37 °C. c d R COO– NH3+ R COO–. Friedman et al. (1965). With SOD/catalase.
NH3+
pKa
SH
S–
Figure 3. Relationship between the hydrogen partial charge of the mercapto group and the rate constants of metHb reduction (k1app) and thiol oxidation (k2app): (4) glutathione (GSH), (0) DLhomocysteine (Hcy), and (O) L-cysteine (Cys).
Figure 6. Time course of the metHb percentage of HbV in vitro (]) at PO2 ) 142 Torr and 37 °C and in vivo after 90 vol % exchange transfusion or 20 vol % overdose into anesthetized Wistar rats ([Hcy] ) 5 mM, [SOD] ) 103 units/mL, and [catalase] ) 105 units/mL): 20 vol % overdose with ([) HbV without Hcy, (O) HbV with Hcy, (4) HbV with Hcy and catalase, and (0) HbV with Hcy and SOD/catalase, and 90 vol % exchange transfusion (b).
Figure 4. Time course of the metHb percentage in an Hb solution. [Hb] ) 6.2 mM, [thiols] ) 10 mM, and [PLP] ) 18.6 mM, in HEPES buffer (50 mM, pH 7.0) with PO2 ) 142 Torr: (O) Cys, (2) Hcy, (b) GSH, (4) Acy, and (0) without thiols. Figure 7. Time course of the metHb percentage of HbV after the stepwise addition of H2O2 ([Hb] ) 0.78 mM, [catalase] ) 105 units/mL, and 3.1 mM of H2O2 was added after 0, 60, 120, and 180 min).
Figure 5. Influence of the concentration of Hcy on the rate of metHb formation in Hb solution ([Hb] ) 5.1 mM, [PLP] ) 15.3 mM, pH 7.4, PO2 ) 142 Torr, and 37 °C): (O) without catalase, (0) with catalase, and (4) with SOD/catalase.
of various reagents without the chemical modification of Hb. It was also confirmed by study in vivo (20% overdose). The rate of metHb of HbV containing 5 mM Hcy was 50% after 24 h, which is 60% lower than that of HbV without Hcy in vivo (83% after 24 h). Therefore, the coencapsulation of Hcy is expected to show high efficiency. When H2O2 was progressively added to the HbV suspension, the metHb percentage increased in response to the addition (Figure 7). H2O2 easily diffuses through
the bilayer membrane and generates hydroxyl radicals by a Fenton reaction in the presence of a trace amount of free iron inside the HbV (Gutteridge, 1986). They convert Hb to metHb. However, the addition of H2O2 to the Hb solution purified by dialysis does not induce the metHb formation because of the absence of free iron or heme. This indicates that, even though the highly purified Hb solution was used for HbV preparation, a small amount of free ion or heme would be released from Hb during the preparation of HbV by extrusion. On the other hand, HbV coencapsulated with catalase did not show such a response to H2O2 addition and maintained a low metHb content (Figure 7), indicating that catalase in HbV diminishes not only H2O2 generated from Hcy oxidation but also H2O2 invading from the outer phase through the bilayer membranes Effect of Hcy and Oxygen Partial Pressure on the MetHb Formation of HbV in Vivo. After the intravenous injection of HbV into rats at a 20 vol % overdose, the rate of metHb formation was about twice as high in comparison with that measured under physiological conditions in vitro at a PO2 of 142 Torr (Figure 6).
MetHb Reduction by Thiols in Hb Vesicles
Bioconjugate Chem., Vol. 8, No. 4, 1997 543 ACKNOWLEDGMENT
This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, and Culture, Japan (07508005 and 08680940), the Ogasawara Foundation for the Promotion of Science and Engineering, and the Kawakami Memorial Foundation. The authors thank Drs. Y. Izumi, A. Yoshizu, and K. Kobayashi (Department of Medicine, Keio University) for their experimental cooperation in animal tests and discussions. LITERATURE CITED Figure 8. PO2 dependence of the initial rate of metHb formation in HbV ([Hb] ) 0.78 mM and pH 7.4).
Furthermore, at a 90% exchange transfusion, the rate was four times as high. Though the coencapsulation of active oxygen scavengers such as SOD and catalase is thought to suppress metHb formation, the coencapsulation had a little effect on metHb suppression in vivo. This indicates that the main reason of the difference in the data between in vitro and in vivo would be due to the ratio of deoxyHb which is oxidized more easily than oxyHb if the appropriate reductant like Hcy is used in the appropriate amount, and the injection amount is 20 vol %. This can be supported by the fact that the addition of H2O2 did not accelerate metHb formation as shown in Figure 7. The influence of PO2 on the rate of metHb formation in HbV was studied and is shown in Figure 8. The rate increases with decreasing PO2. After showing a maximum at a PO2 of 23 Torr, the rate decreases to zero at a PO2 of 0 Torr. It was reported that deoxyHb, to which H2O was weakly coordinated, reacted with oxygen to form metHb more rapidly than did HbO2 (Brantley et al., 1993). When PO2 is higher than 100 Torr, the rate of metHb formation is constant because the degree of oxygen saturation is ca. 100%. The increase in the rate of metHb formation at a PO2 lower than 100 Torr is due to the increase in the amount of the more reactive deoxyHb. The subsequent decrease is due to the lower oxygen concentration which reacts with deoxyHb. The rate at the maximum was almost the same as that in vivo as shown in Figure 6. During blood circulation, HbV is continuously exposed to different PO2s. Usually, PO2 in arterial blood and mixed venous blood is estimated to be 100 and 40 Torr, respectively. That in tissue capillaries is estimated to be 25-30 Torr. Therefore, the reason to accelerate the metHb formation of HbV in vivo would be due to the increased ratio of deoxyHb at a lower PO2. There have been some papers which report the PO2 dependence on the rate of metHb formation in Hb solutions (Levy et al., 1988; Mansouri & Winterhalter, 1973). Of course, active oxygens, including nitric oxide, would cause metHb formation under more severe conditions such as a 90% exchange transfusion or hemorrhagic shock. In this case, coencapsulation of SOD and/or catalase would be effective. From these results, it is apparent that the generation of active oxygens from Hb autoxidation is low when the purity of Hb is high. Because the active oxygens are generated from the autoxidation of reductants, reductants should be selected for a low autoxidation rate. In this sense, Hcy should be the best reductant for HbV. The autoxidation of Hcy in vivo should be slower than that in air because of the low oxygen partial pressure in vivo. Therefore, the higher rate of metHb formation during blood circulation was effectively suppressed by the addition of Hcy.
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BC970091Y
