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Time resolved absorption study of the reaction of hydroxyurea with sickle cell hemoglobin
Biochimica et Biophysica Acta 1380 Ž1998. 64–74
Time resolved absorption study of the reaction of hydroxyurea with sickle cell hemoglobin Daniel B. Kim-Shapiro
a,)
, S. Bruce King b, Challice L. Bonifant b, Christopher P. Kolibash a , Samir K. Ballas c
a
Department of Physics, Wake Forest UniÕersity, Winston-Salem, NC 27109-7507, USA Department of Chemistry, Wake Forest UniÕersity, Winston-Salem, NC 27109-7507, USA The Cardeza Foundation, Department of Medicine, Jefferson Medical College, Philadelphia, PA 19107, USA b
c
Received 14 July 1997; accepted 22 September 1997
Abstract Hydroxyurea has been mixed with hemoglobin S and the reaction was studied using electronic absorption spectroscopy as a function of time and wavelength. The rate of conversion of oxyhemoglobin S to other species was determined and the nature of the reaction products was studied. We also report the formation of methemoglobin Žand other reaction products. when deoxyhemoglobin S is combined with hydroxyurea. The probable increase in the formation of methemoglobin, and other potential reaction products such as nitric oxide-hemoglobin, in patients with sickle cell anemia who are taking hydroxyurea as a therapeutic drug is discussed in terms of the pathophysiology of the disease. It is proposed that methemoglobin and possibly nitric oxide-hemoglobin formation may partially explain beneficial effects observed in these patients before their levels of fetal hemoglobin have increased. q 1998 Elsevier Science B.V. Keywords: Sickle cell hemoglobin; Hydroxyurea; Methemoglobin; Nitric oxide; Time resolved absorption spectroscopy
1. Introduction Sickle cell disease effects about 1 out of 600 people of African descent born in the United States w1x. The disease is caused by a mutant form of hemoglobin, hemoglobin S Ž HbS., which differs from normal adult hemoglobin, hemoglobin A Ž HbA. , by the substitution from L-Glutamate to L-Valine at the b 6 position w2x. The polymerization of HbS, which occurs under hypoxic conditions, causes distortion and increased rigidity of the sickle red blood cell that
)
Corresponding author. Fax: q1 910 759 6142; E-mail: [email protected]
leads to microvascular occlusion and a host of resulting complications w3x. The disease is associated with tissue damage, severe painful crises and a high degree of mortality. A double blind, randomized clinical trial of the use of hydroxyurea as a treatment for sickle cell disease showed that hydroxyurea could reduce the number of painful crises in some patients w4,5x. The beneficial effect of the drug is believed to be largely due to the increase in levels of fetal hemoglobin ŽHbF. , the presence of which reduces the tendency of HbS to polymerize w4,5x. It was noted that some patients benefited from the drug before their levels of HbF increased. Thus it was suggested that there may be other mechanisms in which hydroxyurea affects sickle
0304-4165r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 6 5 Ž 9 7 . 0 0 1 3 2 - 3
D.B. Kim-Shapiro et al.r Biochimica et Biophysica Acta 1380 (1998) 64–74
cell anemia w4,5x. It was proposed that increases in mean cell volume ŽMCV. and cytoreduction Žespecially of neutrophils. associated with hydroxyurea use could have played a role in the beneficial effects of the drug w5x. Increases in MCV would decrease the number of cells in which polymerization occurs. A decrease in neutrophil count could be beneficial since these cells are implicated in contributing to vascular occlusion and tissue damage w5x. Electron Paramagnetic Resonance Ž EPR. studies by K. Stolze and H. Nohl of the reaction of hydoxyurea with oxyhemoglobin Žfrom Bovine red cells. concluded that the oxyhemoglobin was initially converted to methemoglobin ŽMetHb. along with the formation of an aminocarbonylaminooxyl radical ŽH 2 N–CO–NHO . . w6x. It was then proposed that the
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MetHb formed a low spin MetHb-hydoxyurea adduct in a reversible reaction favoring free MetHb. Finally the MetHb-hydroxyurea adduct would then form nitric oxide hemoglobin Ž HbNO., CO 2 and NH 3 w6x. Thus their proposed mechanism suggested that the final fate of the hemoglobin was HbNO. A quantitative analysis of the reaction products was not made. This same group has also recently observed the hydroxyurea mediated conversion of oxymyoglobin to metmyoglobin w7x. In another study, it was found that human erythrocytes that were incubated in hydroxyurea showed elevated levels of methemoglobin formation w8x. In this work we have used absorption spectroscopy to study the reaction of hydroxyurea with oxyHbS. We propose an additional reaction product: hemichromes Ž a form of hemoglobin where
Fig. 1. Time resolved absorption of oxygenated sickle cell hemoglobin in the presence of hydroxyurea. Measurements are shown for 5 min intervals from zero to 14 h after mixing oxyHbS at 240 mM with 660 mM hydroxyurea. Ža. Visible spectra, Žb. Expanded view around isobestic point.
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Table 1 Rate of conversion of oxyHbS Hydroxyurea ŽmM.
Apparent rate constant: OxyHbS Žl sy1 .
330 500 660
5.1=10y5 "2.0=10y5 1.1=10y4 "3.6=10y5 1.5=10y4 "5.3=10y5
the iron is coordinated to an endogenous ligand. . We estimate the percentages of reaction products after the reaction has proceeded to apparent completion. Our evaluation of the percentage of reaction products clearly shows that the main reaction product is methemoglobin and Žcontrary to the implications of ear-
lier work w6x. HbNO is not the predominant reaction product. In addition, we report that hydroxyurea reacts with deoxyHbS to form MetHbS and other species. These findings suggest both beneficial and deleterious affects of the use of hydroxyurea by patients with sickle cell anemia.
2. Materials and methods Excess, discarded blood was obtained that had been drawn from patients homozygous in HbS with low Žless than 5%. fetal hemoglobin following fed-
Fig. 2. Increased turbidity at later times. Ža. Absorption measurements taken every 10 min for 26 h are shown for a mixture of oxyHbS at 200 mM with 500 mM hydroxyurea. Žb. The spectra of the species obtained using global analysis. The spectrum of the starting material, oxyHbS, has peaks at 540 and 577 nm. It was found to decay at a rate of 1.1 = 10y4 Žsy1 . with concomitant formation of the lower of the two other spectra. The third spectra with the displaced baseline appeared at a rate of 1.0 = 10y5 Žsy1 .. The second two spectra represent combinations of species.
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eral regulations and guidelines. Hemoglobin S was prepared as described previously w9x. The cells were washed several times in 0.95% NaCl and then lysed by incubation in distilled water. The membranes were removed by centrifugation and the supernatant was dialyzed against 0.01 M sodium phosphate buffer, pH 7.3. The hemoglobin sample was then pelleted in liquid nitrogen and stored at y808C. Samples used for spectrophotometric measurements were thawed and used within 24 h. All measurements were conducted at room temperature. All solutions were prepared in 0.1 M sodium phosphate buffer, pH 7.3 and all measurements were made in this buffer. HbNO was formed by sequential addition of sodium dithionite Žfinal concentration, 15 mg mly1 . and potassium nitrite Ž final concentration, 5 mg mly1 . to HbS in an argon saturated environment w10,11x. MetHbS was formed by the addition of potassium ferricyanide in a slight molar excess to oxyHbS followed by prolonged dialysis against 0.2 M phosphate buffer Ž pH 6.8. and distilled water w12x. The MetHbS was dialyzed against 0.1 M sodium phosphate buffer at pH 7.3 prior to spectroscopic measurements. Cyanomethemoglobin ŽCNMetHbS. was formed by adding a slight molar
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excess of potassium cyanide to MetHbS w12x. Hemichromes were formed by the addition of sodium salicylate Ž0.5 M final concentration. to MetHbS w13x. DeoxyHbS was prepared by gently flowing argon over oxyHbS for several hours. Final hemoglobin concentrations in kinetic measurements ranged from 0.15 to 0.3 mM. All reactions were conducted in excess HU and no dependence on Hb concentration was observed. In order to scavenge trace amounts of oxygen in measurements involving deoxyHbS, Protocatechuate 3,4-dioxygenase at a final concentration of 0.025 U mly1 and its substrate, Protocatechuic Acid at a final concentration of 200 mM ŽSigma, Saint Louis, Missouri. were added. Hydroxyurea Ž Sigma. was used at concentrations given below. Absorption measurements were performed on an OLIS RSM 1000 Spectrophotometer ŽBogart, Georgia. for stopped-flow measurements, and a PerkinElmer Lambda 9 Ž Norwalk, CT. and a Hewlett Packard 8452A diode array spectrophotometer for measurements conducted on the time scale of hours. The kinetic data were analyzed using Specfit Ž Spectrum Software Associates, Chapel Hill, NC. using singular value decomposition ŽSVD. and global analysis w14,15x. The percentage of pure species in the
Fig. 3. Rapid conversion of MetHbS mixed with 750 mM hydroxyurea. The features at 500 and 630 nm decrease and those at 537 and 575 nm increase with time. One thousand spectral scans per second were taken over 10 s. These were averaged to the 100 curves represented in the figure.
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Table 2 Rates of conversion of MetHbS Hydroxyurea ŽmM.
Apparent rate constant: OxyHbS Žl sy1 .
187.5 375 750
1.30"0.15 1.47"0.051 1.7"0.090
reaction products were determined by a least squares fit to known species normalized by their extinction coefficients w12,13,16,17x.
3. Results The time and spectral dependence of absorption was measured for mixtures of oxyHbS and three different concentrations of hydroxyurea Ž 330, 500 and 660 mM.. Absorption measurements for the reaction of oxyHbS with hydroxyurea at 660 mM are presented in Fig. 1Ž a.. Data was collected every ten minutes over 12 h. The conversion mainly to MetHbS is indicated by the growing absorbance at 635 and 500 nm and the decreasing absorbance peaks of the oxyHbS spectrum Ž at 540 and 577 nm. . Similar measurements using urea instead of hydroxyurea did not produce these absorption changes. Fig. 1Ž b. provides an expanded view around apparent isobestic points
for the reaction shown in Fig. 1Ž a. . Six measurements were made at each concentration. The data were analyzed by SVD and global analysis fitting to a single exponential process. The average of the apparent rate constants obtained for reactions at each concentration of hydroxyurea for oxyHbS are shown in Table 1. Each apparent rate constant is shown " one standard deviation. The largest residual Ž the difference between the absorbance predicted by the fit and the actual measured data. for any wavelength at any time was 0.01 OD for the data shown in Fig. 1Ža.. When the reaction was followed for a longer time a small change in the spectra is noted along with an increase in turbidity ŽFig. 2Ž a... The increased turbidity is reminiscent of hemichrome formation w18x. Hemichromes are normal products of methemoglobin on the path of denaturation that leads to aggregation and increased turbidity. The formation of hemichromes was also supported by decreased helicity measured by circular dichroism Ž data not shown. . Global analysis of these data, fit to two exponential processes, revealed spectra represented in Fig. 2Ž b. with rate constants of 1.1 = 10y4 and 1.0 = 10y5 Žsy1 .. Working with Bovine hemoglobin, Stolze and Nohl proposed that MetHb Žformed in the reaction of HU with oxyHb. quickly forms a MetHb-HU adduct with the equilibrium favoring free MetHb. Fig. 3 shows
Fig. 4. Conversion of deoxyHbS. Measurements are shown for 8 min intervals from zero to 120 min after mixing deoxyHbS at 180 mM with 260 mM hydroxyurea.
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the time resolved absorption from rapid mixing of HU with previously prepared MetHbS. The reaction proceeded rapidly with an observed rate of 1.5 lŽ sy1 ..
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Rapid mixing under identical concentrations of urea did not produce any spectral changes on these time scales. The mixture underwent additional, slower re-
Fig. 5. Determination of fraction of species in the product. Ža. The millimolar extinction profiles Ždivided by 10. of the components used in the fit: HbSNO Žhaving two equal peaks at 545 and 573 nm., CNMetHbS Žhaving a single peak at 535 nm., hemichrome Žpeaking at 536 nm with a shoulder at 575 nm., and scattering Žshowing a 1rŽ l4 . dependence.. Žb. Comparison of data and theoretical fit using the curves in Fig. 5Ža.. The two curves mostly overlap except around 610 nm where the theoretical fit is slightly lower than the measurement. The fit is composed of 0.096 mM CNMetHbS, 0.012 mM hemichromes, 0.018 mM HbS-NO and 0.02 times the scattering curve. Žc. Comparison of data and theoretical fit using the curves in Fig. 5Ža. except for that of HbNO. The theoretical fit is blue-shifted compared to the data. The fit is composed of 0.1 mM CNMetHbS curve, 0.032 mM hemichromes, and 0.01 times the scattering curve.
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actions resulting in absorption spectra like that finally obtained after reacting Hu and oxyHbS. Table 2 shows the average of seven mixtures at three different concentrations. The amplitude of the difference spectra between the product and reactant species decreased with increasing HU concentration. In order to assess the ramifications of the reaction of hydroxyurea and HbS in vivo it is important to know if there is any reaction with deoxyHbS. Fig. 4 shows Žfor the first time. time resolved absorption measurements for this reaction. Apparently the major product is methemoglobin but the lack of a tight isobestic point indicates that other species are present even before the first reaction is complete. The average rate of conversion of deoxyHbS from mixtures at three concentrations is Ž8.08 " 1.63. = 10y4 ŽMsy1 .. In order to assess the percentage of different components resulting from the mixture of HU with oxyHbS, we used a least square analysis to decompose the spectra of the product mixture combined with potassium cyanide to known species. The resulting spectrum was fit to the species shown in Fig. 5Ž a.. A comparison of the data and a fit to spectra of CNmetHb, hemichromes, HbNO and scattering are shown in Fig. 5Žb.. Fitting 6 product mixtures reacted with potassium cyanide resulted in an estimate of 77 " 6% CNMetHb, 13 " 3% hemichromes and 10 " 7% HbNO. Fig. 5Ž c. shows a comparison of the data and a fit to spectra of CNmetHb, hemichromes, and scattering. Fitting 6 product mixtures reacted with potassium cyanide resulted in an estimate of 80 " 8% CNMetHb and 20 " 8% hemichromes. For each fit we calculated the residuals Ž the sum of the squares of the difference of the data and the theoretical fit for 225 wavelengths.. The average of the residuals for the fits that included HbNO was 0.033 and for those that did not include HbNO was 0.090.
4. Discussion The time resolved absorption spectra shown in Fig. 1Ža. demonstrates the conversion of oxyHbS to other species in the presence of HU. The resulting product spectra resembles MetHbS but are not identical with pure MetHbS absorption. This fact, taken together with the presence of isobestic points, suggests that oxyHbS is converted to MetHbS which can then
partake in another relatively fast reaction. This latter reaction is probably the same of that of pure MetHbS with HU Ž Fig. 3.. The concentration dependence of this fast reaction suggests that MetHbS combines with HU to form another species in a reversible reaction so that an equilibrium mixture of MetHbS and this other species Žpossibly the MetHb-HU adduct proposed by Stolze and Nohl w6x. is rapidly obtained. The rate of backward reaction dominates the observed rate and free MetHbS is favored. Thus our data suggest that k1
oxyHbSq HU ™ MetHbSq HU derivative; k 2f
MetHbSq HU ° MetHbSy HU, k 2b
Ž1.
where k 2b dominates the second reaction. The presence of the MetHbS adduct is based on previous assignment w6x and requires confirmation. Since the second reaction is so much faster than the first, an isobestic is observed that corresponds to where oxyHbS and the rapidly obtained equilibrium mixture of MetHbS and the product of the second reaction have the same extinction coefficient. The rates given in Table 1 correspond to k 1. We suggest that this reaction follows pseudo first order kinetics. Although the relation between the average observed rate and the concentration of HU is not linear, a linear relationship does describe the observed rates along with their standard deviations. At this time, pseudo first order kinetics is the best description that we can provide for the first reaction. The average value for k 1 is Ž2.00 " 0.40. = 10y4 l sy1 ŽM... We attribute the increased turbidity demonstrated in Fig. 2Ža. to hemichrome formation, followed by denaturation and aggregation. The formation of hemichromes on this time scale is faster than that observed for normal adult hemoglobin Ž HbA. w18x. This could be due to HbS being less stable than HbA and due to the presence of the HU which might interfere with hydrogen bonding causing faster denaturation. We did not use a spectrum of the final reaction mixture to fit to known species and determine their relative abundances because we do not know the spectrum of one possible species, the HbS-HU adduct.
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One may be tempted to use the spectral measurements from the reaction of MetHbS with HU Ž Fig. 3. to obtain a spectrum for the HbS-HU adduct. However, it would be difficult to separate this spectrum from MetHbS, which is favored in equilibrium in this reaction. Furthermore, we cannot know that this reaction does not involve more than the two species ŽMetHbS and the MetHbS-HU adduct. such as HbNO. Thus we use the reaction mixture reacted with potassium cyanide ŽKCN. . KCN will not react with HbNO but will form CNMetHb from MetHb and should displace another weaker ligand such as HU. The fraction of CNMetHbS Ž 77%. could be partially from HbS-HU where the HU is displaced. It is surprising that 13% of the products were found to be hemichromes since CN is known to sometimes reverse hemichrome formation w13x. The types of hemichromes that were present may thus have included irreversible types w12x. Deviations in the spectra of these types of hemichromes from those that we made using salicylate may partially account for deviations of the theoretical fits from the observed data. Our data suggest that some HbNO is present. A comparison of Fig. 5Ž b. and Ž c. indicates how the inclusion of HbNO improves our fits but it should be remembered that an additional parameter is bound to improve the fit. One of our major findings in this study is that HbNO is not the major reaction product in the reaction of HbS and HU as implicated previously w6x. We have shown that deoxyHbS is also converted to other forms in the presence of HU Ž Fig. 4. . MetHbS appears to be the primary product in this reaction. The rate of conversion of deoxyHbS is faster than that of oxyHbS by a factor of four. There appears to be more than two species present even during the early stages of the reaction involving deoxyHbS. The nature of these species is currently under investigation. Here we want to emphasize that one would expect conversion of HbS in the presence of HU during both the venous and arterial circulation. Mechanistic details of the reactions between hydroxyurea and various heme proteins remain poorly understood. Stolze and Nohl proposed that the hydroxyurea mediated methemoglobin formation from oxyhemoglobin occurs through the heme bound oxygen cooxidation of the heme iron Ž Feq2 to Feq3 . and hydroxyurea to the aminocarbonylaminooxyl radical
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ŽH 2 N–CO–NHO . . which has been identified by EPR spectroscopy w6x. Recent EPR spectroscopic studies indicate that this radical, when generated from hydroxyurea in the presence of hemoglobin and hydrogen peroxide, forms nitric oxide Ž NO. w19x, which is known to react with various heme proteins w20x. Treatment of hydroxyurea with hydrogen peroxide and copper ŽII. sulfate also produces an ‘‘NO like’’ product w21x. Taken together, these results suggest that some of the observed reactions of hydroxyurea with heme proteins could be mediated through nitric oxide or a similar nitrogen oxide such as nitroxyl ŽHNO or yNO. w22–24x. The conversion to MetHbS is not likely to be due simply to an increase in an autooxidation rate that results from the loosening of the protein structure by hydroxyurea. It might be suggested that, although hydroxyurea does not denature the protein at the concentrations used in this investigation, it might interfere with hydrogen bonding enough to allow faster autooxidation. Such a notion is not consistent with measurements made in the presence of 260 mM urea where we observed no significant conversion to MetHbS. To assess the ramifications of the conversion to MetHbS by hydroxyurea in patients taking this drug we must consider the concentrations of hydroxyurea and hemoglobin used in this in vitro study and those found in patients. Levels of hydroxyurea in the plasma of patients undergoing this therapy are about 0.3 mM w25,26x. The concentration of hydroxyurea in the red blood cell equilibrates quickly Ž within several seconds. to that in the plasma w27x. In the red cell, the bimolecular reaction involving HU and HbS would be governed by the HbS concentration. Preliminary observations in our lab indicate that the rate of HbS conversion increases by a factor of two when the temperature is increased by 108C. If we take the concentration of HbS in the red blood cell to be 20 mM in heme then Žusing an average bimolecular rate constant for deoxyHbS and oxyHbS at of 1.0 = 10y3 l sy1 .ŽM... we calculate a rate of disappearance of hydroxyurea Ž and concomitant rate of HbS conversion. of 2.0 = 10y5 per second. Assuming this rate would be unaffected by other factors in the cell, it corresponds to 0.13 mM of HbS converted per day. This extremely rough calculation suffers from having ignored other effects on MetHbS formation that would be present in the sickle red blood cell. In the red cell
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the rate of MetHbS formation is complicated by the presence of reducing agents and other oxidizing agents. Our rough calculation of MetHbS formation in vivo is considerably less than what was found in a study where levels of MetHb increased by approximately 0.8 mM Žwith respect to the control. in red cells incubated over 24 h in 0.1 mM hydroxyurea w8x. It is possible that the presence of other oxidants in the red cell could accelerate the reaction between hydroxyurea and hemoglobin. A recent report indicates the participation of hydrogen peroxide Ž found in the sickle red blood cell in elevated levels w28x. in the reaction of hydroxyurea and hemoglobin w19x. Preliminary observations in our laboratories show that the presence of hydrogen peroxide and hydroxyurea results in MetHb formation at a faster rate than the sum of the rates that would be obtained with only one of these reagents present at a time ŽFig. 6.. Although some MetHbS that is formed could be reduced to deoxyHbS in the cell, some of it will take different irreversible pathways and the decrease in oxyHbS and deoxyHbS concentration could be an affect that accumulates over the lifetime of the red cell. An increase in MetHb formation in sickle cell patients would have several negative ramifications. There are many deleterious affects associated with MetHb formation in sickle cell patients Ž for a review
Fig. 6. Time resolved absorption of sickle cell hemoglobin Ž228 mM. and hydroxyurea Ž66 mM. in the presence of hydrogen peroxide Ž2.2 mM.. Seven absorption measurements are shown from 3 ms after mixing to 180 s after mixing, evenly spaced 30 s apart.
see w29x or w30x.. Sickle cell patients have elevated levels of MetHbS due to a higher autooxidation rate of HbS and a lower MetHb reduction rate w29x. The increased levels of MetHb result in increased hemichrome formation, heme loss, and heme associated membranes. These effects together with the increase in oxidants that result from MetHbS formation lead to membrane damage characterized by decreased stability and increased rigidity w28x. A previous study by Ballas et al. studied the rheological properties of red cells from two patients as they underwent hydroxyurea therapy w31x. Both patients responded positively to the treatment with respect to an increase in HbF levels. It was found that the deformability properties of the cells of these patients improved and the membrane stability remained normal. The onset of the improvement of rheological properties associated with treatment with hydroxyurea did not correlate perfectly with an increase in HbF levels w31x. Thus it was suggested that another mechanism may contribute to improvement in red cell rheological properties for patients taking hydroxyurea w31x. In another study, it was found that normal red cells that were incubated in hydroxyurea experienced membrane damage and became more fragile w8x. These results, taken together suggest that the beneficial rheological effects observed by Ballas et al. in vivo resulted from reduced HbS polymerization. The positive effect of MetHbS formation, discussed below, may have played a role. The main positive effect of MetHbS formation for sickle cell patients would be that the tendency for HbS polymerization would decrease as a result of a decreased cellular concentration of deoxyHbS during circulation w30,32x. This would also be the case for HbS converted to the HbNO form. In fact, increasing the level of MetHbS and HbNO have been considered as a treatment for sickle cell disease w30,33x. Even a small change in the concentration of deoxyHbS can have a large effect on whether HbS polymerization occurs because of the effect on the kinetics of polymerization. HbS polymerization is characterized by a long period of time where no polymerization Žknown as the delay time. occurs followed by rapid exponential polymerization w34–37x. Many red blood cells escape HbS polymerization by reaching the venous circulation or being reoxygenated before polymerization begins w38x. The delay time is
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roughly related to the concentration of deoxyHbS by the relation w35x 1 c0 n sl td cs
ž /
where td is the delay time, c 0 is the total HbS concentration, cs is the solubility Žthat is the amount of deoxyHbS that will remain in the solution Žas opposed to the polymer. phase. , is a proportionality factor and n is between 30 and 40. This means that if the concentration of deoxyHbS decreases to 19.7 from 20 mM the delay time Žwith n s 40. becomes 1.8 times longer. Although we have ignored any small changes that would occur involving the solubility of deoxyHbS Žfrom non-ideality considerations. , it is certain that such a change in the cellular concentrations of deoxyHbS due to MetHbS formation would cause large changes in td thus preventing many cells from becoming rigid and contributing to microvascular occlusion. We hypothesize that the beneficial affect that some patients undergoing hydroxyurea therapy experience is due, in part, to MetHbS and possibly HbS-NO formation. Thus MetHbS and HbSNO formation could help to explain improvements in rheological properties w31x and clinical manifestations w4,5x observed in patients taking hydroxyurea. Acknowledgements We thank Yiren Gu for helpful discussion. This work was supported by Wake Forest University startup funds ŽDBK-S., the American Heart Association Žgrant 963031N awarded to SBK. and the National Institutes of Health Comprehensive Sickle Cell Center Grant HL38632 awarded to SKB. References w1x D.L. Rucknagel, in: R.D. Levere ŽEd.., Sickle Cell Anemia and Other Hemoglobinopathies, Academic Press, New York, 1975, p. 1. w2x V.M. Ingram, Nature 178 Ž1956. 792–794. w3x S.H. Embury, R.P. Hebbel, N. Mohandas, M.H. Steinberg, Sickle Cell Disease. Basic Principles and Clinical Practice, Raven Press, New York, 1994. w4x S. Charache, M.L. Terrin, R.D. Moore, G.J. Dover, F.B. Barton, S.V. Eckert, R.P. McMahon, D.R. Bonds, Investigators of the multicenter study of hydroxyurea in sickle cell anemia, New Engl. J. Med. 332 Ž1995. 1317–1322.
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w31x S.K. Ballas, G.J. Dover, S. Charache, Am. J. Hematol. 32 Ž1989. 104–111. w32x H.A. Itano, Sickle Cell Anemia, Ph. D. Thesis, California Institute of Technology, 1950. w33x W.A. Mcdade, H.M. Shaba, N. Carter, Biophys. J. 72 Ž1996. A9. w34x J. Hofrichter, P.D. Ross, W.A. Eaton, Proc. Natl. Acad. Sci. U.S.A. 71 Ž1974. 4864–4868.
w35x J. Hofrichter, P.D. Ross, W.A. Eaton, Proc. Natl. Acad. Sci. U.S.A. 73 Ž1976. 3034–3039. w36x F.A. Ferrone, J. Hofrichter, W.A. Eaton, J. Mol. Biol. 183 Ž1985. 591–610. w37x F.A. Ferrone, J. Hofrichter, W.A. Earon, J. Mol. Biol. 183 Ž1985. 611–631. w38x A. Mozarelli, J. Hofrichter, W.A. Eaton, Science 237 Ž1987. 500–506.
Time resolved absorption study of the reaction of hydroxyurea with sickle cell hemoglobin Daniel B. Kim-Shapiro
a,)
, S. Bruce King b, Challice L. Bonifant b, Christopher P. Kolibash a , Samir K. Ballas c
a
Department of Physics, Wake Forest UniÕersity, Winston-Salem, NC 27109-7507, USA Department of Chemistry, Wake Forest UniÕersity, Winston-Salem, NC 27109-7507, USA The Cardeza Foundation, Department of Medicine, Jefferson Medical College, Philadelphia, PA 19107, USA b
c
Received 14 July 1997; accepted 22 September 1997
Abstract Hydroxyurea has been mixed with hemoglobin S and the reaction was studied using electronic absorption spectroscopy as a function of time and wavelength. The rate of conversion of oxyhemoglobin S to other species was determined and the nature of the reaction products was studied. We also report the formation of methemoglobin Žand other reaction products. when deoxyhemoglobin S is combined with hydroxyurea. The probable increase in the formation of methemoglobin, and other potential reaction products such as nitric oxide-hemoglobin, in patients with sickle cell anemia who are taking hydroxyurea as a therapeutic drug is discussed in terms of the pathophysiology of the disease. It is proposed that methemoglobin and possibly nitric oxide-hemoglobin formation may partially explain beneficial effects observed in these patients before their levels of fetal hemoglobin have increased. q 1998 Elsevier Science B.V. Keywords: Sickle cell hemoglobin; Hydroxyurea; Methemoglobin; Nitric oxide; Time resolved absorption spectroscopy
1. Introduction Sickle cell disease effects about 1 out of 600 people of African descent born in the United States w1x. The disease is caused by a mutant form of hemoglobin, hemoglobin S Ž HbS., which differs from normal adult hemoglobin, hemoglobin A Ž HbA. , by the substitution from L-Glutamate to L-Valine at the b 6 position w2x. The polymerization of HbS, which occurs under hypoxic conditions, causes distortion and increased rigidity of the sickle red blood cell that
)
Corresponding author. Fax: q1 910 759 6142; E-mail: [email protected]
leads to microvascular occlusion and a host of resulting complications w3x. The disease is associated with tissue damage, severe painful crises and a high degree of mortality. A double blind, randomized clinical trial of the use of hydroxyurea as a treatment for sickle cell disease showed that hydroxyurea could reduce the number of painful crises in some patients w4,5x. The beneficial effect of the drug is believed to be largely due to the increase in levels of fetal hemoglobin ŽHbF. , the presence of which reduces the tendency of HbS to polymerize w4,5x. It was noted that some patients benefited from the drug before their levels of HbF increased. Thus it was suggested that there may be other mechanisms in which hydroxyurea affects sickle
0304-4165r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 6 5 Ž 9 7 . 0 0 1 3 2 - 3
D.B. Kim-Shapiro et al.r Biochimica et Biophysica Acta 1380 (1998) 64–74
cell anemia w4,5x. It was proposed that increases in mean cell volume ŽMCV. and cytoreduction Žespecially of neutrophils. associated with hydroxyurea use could have played a role in the beneficial effects of the drug w5x. Increases in MCV would decrease the number of cells in which polymerization occurs. A decrease in neutrophil count could be beneficial since these cells are implicated in contributing to vascular occlusion and tissue damage w5x. Electron Paramagnetic Resonance Ž EPR. studies by K. Stolze and H. Nohl of the reaction of hydoxyurea with oxyhemoglobin Žfrom Bovine red cells. concluded that the oxyhemoglobin was initially converted to methemoglobin ŽMetHb. along with the formation of an aminocarbonylaminooxyl radical ŽH 2 N–CO–NHO . . w6x. It was then proposed that the
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MetHb formed a low spin MetHb-hydoxyurea adduct in a reversible reaction favoring free MetHb. Finally the MetHb-hydroxyurea adduct would then form nitric oxide hemoglobin Ž HbNO., CO 2 and NH 3 w6x. Thus their proposed mechanism suggested that the final fate of the hemoglobin was HbNO. A quantitative analysis of the reaction products was not made. This same group has also recently observed the hydroxyurea mediated conversion of oxymyoglobin to metmyoglobin w7x. In another study, it was found that human erythrocytes that were incubated in hydroxyurea showed elevated levels of methemoglobin formation w8x. In this work we have used absorption spectroscopy to study the reaction of hydroxyurea with oxyHbS. We propose an additional reaction product: hemichromes Ž a form of hemoglobin where
Fig. 1. Time resolved absorption of oxygenated sickle cell hemoglobin in the presence of hydroxyurea. Measurements are shown for 5 min intervals from zero to 14 h after mixing oxyHbS at 240 mM with 660 mM hydroxyurea. Ža. Visible spectra, Žb. Expanded view around isobestic point.
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Table 1 Rate of conversion of oxyHbS Hydroxyurea ŽmM.
Apparent rate constant: OxyHbS Žl sy1 .
330 500 660
5.1=10y5 "2.0=10y5 1.1=10y4 "3.6=10y5 1.5=10y4 "5.3=10y5
the iron is coordinated to an endogenous ligand. . We estimate the percentages of reaction products after the reaction has proceeded to apparent completion. Our evaluation of the percentage of reaction products clearly shows that the main reaction product is methemoglobin and Žcontrary to the implications of ear-
lier work w6x. HbNO is not the predominant reaction product. In addition, we report that hydroxyurea reacts with deoxyHbS to form MetHbS and other species. These findings suggest both beneficial and deleterious affects of the use of hydroxyurea by patients with sickle cell anemia.
2. Materials and methods Excess, discarded blood was obtained that had been drawn from patients homozygous in HbS with low Žless than 5%. fetal hemoglobin following fed-
Fig. 2. Increased turbidity at later times. Ža. Absorption measurements taken every 10 min for 26 h are shown for a mixture of oxyHbS at 200 mM with 500 mM hydroxyurea. Žb. The spectra of the species obtained using global analysis. The spectrum of the starting material, oxyHbS, has peaks at 540 and 577 nm. It was found to decay at a rate of 1.1 = 10y4 Žsy1 . with concomitant formation of the lower of the two other spectra. The third spectra with the displaced baseline appeared at a rate of 1.0 = 10y5 Žsy1 .. The second two spectra represent combinations of species.
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eral regulations and guidelines. Hemoglobin S was prepared as described previously w9x. The cells were washed several times in 0.95% NaCl and then lysed by incubation in distilled water. The membranes were removed by centrifugation and the supernatant was dialyzed against 0.01 M sodium phosphate buffer, pH 7.3. The hemoglobin sample was then pelleted in liquid nitrogen and stored at y808C. Samples used for spectrophotometric measurements were thawed and used within 24 h. All measurements were conducted at room temperature. All solutions were prepared in 0.1 M sodium phosphate buffer, pH 7.3 and all measurements were made in this buffer. HbNO was formed by sequential addition of sodium dithionite Žfinal concentration, 15 mg mly1 . and potassium nitrite Ž final concentration, 5 mg mly1 . to HbS in an argon saturated environment w10,11x. MetHbS was formed by the addition of potassium ferricyanide in a slight molar excess to oxyHbS followed by prolonged dialysis against 0.2 M phosphate buffer Ž pH 6.8. and distilled water w12x. The MetHbS was dialyzed against 0.1 M sodium phosphate buffer at pH 7.3 prior to spectroscopic measurements. Cyanomethemoglobin ŽCNMetHbS. was formed by adding a slight molar
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excess of potassium cyanide to MetHbS w12x. Hemichromes were formed by the addition of sodium salicylate Ž0.5 M final concentration. to MetHbS w13x. DeoxyHbS was prepared by gently flowing argon over oxyHbS for several hours. Final hemoglobin concentrations in kinetic measurements ranged from 0.15 to 0.3 mM. All reactions were conducted in excess HU and no dependence on Hb concentration was observed. In order to scavenge trace amounts of oxygen in measurements involving deoxyHbS, Protocatechuate 3,4-dioxygenase at a final concentration of 0.025 U mly1 and its substrate, Protocatechuic Acid at a final concentration of 200 mM ŽSigma, Saint Louis, Missouri. were added. Hydroxyurea Ž Sigma. was used at concentrations given below. Absorption measurements were performed on an OLIS RSM 1000 Spectrophotometer ŽBogart, Georgia. for stopped-flow measurements, and a PerkinElmer Lambda 9 Ž Norwalk, CT. and a Hewlett Packard 8452A diode array spectrophotometer for measurements conducted on the time scale of hours. The kinetic data were analyzed using Specfit Ž Spectrum Software Associates, Chapel Hill, NC. using singular value decomposition ŽSVD. and global analysis w14,15x. The percentage of pure species in the
Fig. 3. Rapid conversion of MetHbS mixed with 750 mM hydroxyurea. The features at 500 and 630 nm decrease and those at 537 and 575 nm increase with time. One thousand spectral scans per second were taken over 10 s. These were averaged to the 100 curves represented in the figure.
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Table 2 Rates of conversion of MetHbS Hydroxyurea ŽmM.
Apparent rate constant: OxyHbS Žl sy1 .
187.5 375 750
1.30"0.15 1.47"0.051 1.7"0.090
reaction products were determined by a least squares fit to known species normalized by their extinction coefficients w12,13,16,17x.
3. Results The time and spectral dependence of absorption was measured for mixtures of oxyHbS and three different concentrations of hydroxyurea Ž 330, 500 and 660 mM.. Absorption measurements for the reaction of oxyHbS with hydroxyurea at 660 mM are presented in Fig. 1Ž a.. Data was collected every ten minutes over 12 h. The conversion mainly to MetHbS is indicated by the growing absorbance at 635 and 500 nm and the decreasing absorbance peaks of the oxyHbS spectrum Ž at 540 and 577 nm. . Similar measurements using urea instead of hydroxyurea did not produce these absorption changes. Fig. 1Ž b. provides an expanded view around apparent isobestic points
for the reaction shown in Fig. 1Ž a. . Six measurements were made at each concentration. The data were analyzed by SVD and global analysis fitting to a single exponential process. The average of the apparent rate constants obtained for reactions at each concentration of hydroxyurea for oxyHbS are shown in Table 1. Each apparent rate constant is shown " one standard deviation. The largest residual Ž the difference between the absorbance predicted by the fit and the actual measured data. for any wavelength at any time was 0.01 OD for the data shown in Fig. 1Ža.. When the reaction was followed for a longer time a small change in the spectra is noted along with an increase in turbidity ŽFig. 2Ž a... The increased turbidity is reminiscent of hemichrome formation w18x. Hemichromes are normal products of methemoglobin on the path of denaturation that leads to aggregation and increased turbidity. The formation of hemichromes was also supported by decreased helicity measured by circular dichroism Ž data not shown. . Global analysis of these data, fit to two exponential processes, revealed spectra represented in Fig. 2Ž b. with rate constants of 1.1 = 10y4 and 1.0 = 10y5 Žsy1 .. Working with Bovine hemoglobin, Stolze and Nohl proposed that MetHb Žformed in the reaction of HU with oxyHb. quickly forms a MetHb-HU adduct with the equilibrium favoring free MetHb. Fig. 3 shows
Fig. 4. Conversion of deoxyHbS. Measurements are shown for 8 min intervals from zero to 120 min after mixing deoxyHbS at 180 mM with 260 mM hydroxyurea.
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the time resolved absorption from rapid mixing of HU with previously prepared MetHbS. The reaction proceeded rapidly with an observed rate of 1.5 lŽ sy1 ..
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Rapid mixing under identical concentrations of urea did not produce any spectral changes on these time scales. The mixture underwent additional, slower re-
Fig. 5. Determination of fraction of species in the product. Ža. The millimolar extinction profiles Ždivided by 10. of the components used in the fit: HbSNO Žhaving two equal peaks at 545 and 573 nm., CNMetHbS Žhaving a single peak at 535 nm., hemichrome Žpeaking at 536 nm with a shoulder at 575 nm., and scattering Žshowing a 1rŽ l4 . dependence.. Žb. Comparison of data and theoretical fit using the curves in Fig. 5Ža.. The two curves mostly overlap except around 610 nm where the theoretical fit is slightly lower than the measurement. The fit is composed of 0.096 mM CNMetHbS, 0.012 mM hemichromes, 0.018 mM HbS-NO and 0.02 times the scattering curve. Žc. Comparison of data and theoretical fit using the curves in Fig. 5Ža. except for that of HbNO. The theoretical fit is blue-shifted compared to the data. The fit is composed of 0.1 mM CNMetHbS curve, 0.032 mM hemichromes, and 0.01 times the scattering curve.
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actions resulting in absorption spectra like that finally obtained after reacting Hu and oxyHbS. Table 2 shows the average of seven mixtures at three different concentrations. The amplitude of the difference spectra between the product and reactant species decreased with increasing HU concentration. In order to assess the ramifications of the reaction of hydroxyurea and HbS in vivo it is important to know if there is any reaction with deoxyHbS. Fig. 4 shows Žfor the first time. time resolved absorption measurements for this reaction. Apparently the major product is methemoglobin but the lack of a tight isobestic point indicates that other species are present even before the first reaction is complete. The average rate of conversion of deoxyHbS from mixtures at three concentrations is Ž8.08 " 1.63. = 10y4 ŽMsy1 .. In order to assess the percentage of different components resulting from the mixture of HU with oxyHbS, we used a least square analysis to decompose the spectra of the product mixture combined with potassium cyanide to known species. The resulting spectrum was fit to the species shown in Fig. 5Ž a.. A comparison of the data and a fit to spectra of CNmetHb, hemichromes, HbNO and scattering are shown in Fig. 5Žb.. Fitting 6 product mixtures reacted with potassium cyanide resulted in an estimate of 77 " 6% CNMetHb, 13 " 3% hemichromes and 10 " 7% HbNO. Fig. 5Ž c. shows a comparison of the data and a fit to spectra of CNmetHb, hemichromes, and scattering. Fitting 6 product mixtures reacted with potassium cyanide resulted in an estimate of 80 " 8% CNMetHb and 20 " 8% hemichromes. For each fit we calculated the residuals Ž the sum of the squares of the difference of the data and the theoretical fit for 225 wavelengths.. The average of the residuals for the fits that included HbNO was 0.033 and for those that did not include HbNO was 0.090.
4. Discussion The time resolved absorption spectra shown in Fig. 1Ža. demonstrates the conversion of oxyHbS to other species in the presence of HU. The resulting product spectra resembles MetHbS but are not identical with pure MetHbS absorption. This fact, taken together with the presence of isobestic points, suggests that oxyHbS is converted to MetHbS which can then
partake in another relatively fast reaction. This latter reaction is probably the same of that of pure MetHbS with HU Ž Fig. 3.. The concentration dependence of this fast reaction suggests that MetHbS combines with HU to form another species in a reversible reaction so that an equilibrium mixture of MetHbS and this other species Žpossibly the MetHb-HU adduct proposed by Stolze and Nohl w6x. is rapidly obtained. The rate of backward reaction dominates the observed rate and free MetHbS is favored. Thus our data suggest that k1
oxyHbSq HU ™ MetHbSq HU derivative; k 2f
MetHbSq HU ° MetHbSy HU, k 2b
Ž1.
where k 2b dominates the second reaction. The presence of the MetHbS adduct is based on previous assignment w6x and requires confirmation. Since the second reaction is so much faster than the first, an isobestic is observed that corresponds to where oxyHbS and the rapidly obtained equilibrium mixture of MetHbS and the product of the second reaction have the same extinction coefficient. The rates given in Table 1 correspond to k 1. We suggest that this reaction follows pseudo first order kinetics. Although the relation between the average observed rate and the concentration of HU is not linear, a linear relationship does describe the observed rates along with their standard deviations. At this time, pseudo first order kinetics is the best description that we can provide for the first reaction. The average value for k 1 is Ž2.00 " 0.40. = 10y4 l sy1 ŽM... We attribute the increased turbidity demonstrated in Fig. 2Ža. to hemichrome formation, followed by denaturation and aggregation. The formation of hemichromes on this time scale is faster than that observed for normal adult hemoglobin Ž HbA. w18x. This could be due to HbS being less stable than HbA and due to the presence of the HU which might interfere with hydrogen bonding causing faster denaturation. We did not use a spectrum of the final reaction mixture to fit to known species and determine their relative abundances because we do not know the spectrum of one possible species, the HbS-HU adduct.
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One may be tempted to use the spectral measurements from the reaction of MetHbS with HU Ž Fig. 3. to obtain a spectrum for the HbS-HU adduct. However, it would be difficult to separate this spectrum from MetHbS, which is favored in equilibrium in this reaction. Furthermore, we cannot know that this reaction does not involve more than the two species ŽMetHbS and the MetHbS-HU adduct. such as HbNO. Thus we use the reaction mixture reacted with potassium cyanide ŽKCN. . KCN will not react with HbNO but will form CNMetHb from MetHb and should displace another weaker ligand such as HU. The fraction of CNMetHbS Ž 77%. could be partially from HbS-HU where the HU is displaced. It is surprising that 13% of the products were found to be hemichromes since CN is known to sometimes reverse hemichrome formation w13x. The types of hemichromes that were present may thus have included irreversible types w12x. Deviations in the spectra of these types of hemichromes from those that we made using salicylate may partially account for deviations of the theoretical fits from the observed data. Our data suggest that some HbNO is present. A comparison of Fig. 5Ž b. and Ž c. indicates how the inclusion of HbNO improves our fits but it should be remembered that an additional parameter is bound to improve the fit. One of our major findings in this study is that HbNO is not the major reaction product in the reaction of HbS and HU as implicated previously w6x. We have shown that deoxyHbS is also converted to other forms in the presence of HU Ž Fig. 4. . MetHbS appears to be the primary product in this reaction. The rate of conversion of deoxyHbS is faster than that of oxyHbS by a factor of four. There appears to be more than two species present even during the early stages of the reaction involving deoxyHbS. The nature of these species is currently under investigation. Here we want to emphasize that one would expect conversion of HbS in the presence of HU during both the venous and arterial circulation. Mechanistic details of the reactions between hydroxyurea and various heme proteins remain poorly understood. Stolze and Nohl proposed that the hydroxyurea mediated methemoglobin formation from oxyhemoglobin occurs through the heme bound oxygen cooxidation of the heme iron Ž Feq2 to Feq3 . and hydroxyurea to the aminocarbonylaminooxyl radical
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ŽH 2 N–CO–NHO . . which has been identified by EPR spectroscopy w6x. Recent EPR spectroscopic studies indicate that this radical, when generated from hydroxyurea in the presence of hemoglobin and hydrogen peroxide, forms nitric oxide Ž NO. w19x, which is known to react with various heme proteins w20x. Treatment of hydroxyurea with hydrogen peroxide and copper ŽII. sulfate also produces an ‘‘NO like’’ product w21x. Taken together, these results suggest that some of the observed reactions of hydroxyurea with heme proteins could be mediated through nitric oxide or a similar nitrogen oxide such as nitroxyl ŽHNO or yNO. w22–24x. The conversion to MetHbS is not likely to be due simply to an increase in an autooxidation rate that results from the loosening of the protein structure by hydroxyurea. It might be suggested that, although hydroxyurea does not denature the protein at the concentrations used in this investigation, it might interfere with hydrogen bonding enough to allow faster autooxidation. Such a notion is not consistent with measurements made in the presence of 260 mM urea where we observed no significant conversion to MetHbS. To assess the ramifications of the conversion to MetHbS by hydroxyurea in patients taking this drug we must consider the concentrations of hydroxyurea and hemoglobin used in this in vitro study and those found in patients. Levels of hydroxyurea in the plasma of patients undergoing this therapy are about 0.3 mM w25,26x. The concentration of hydroxyurea in the red blood cell equilibrates quickly Ž within several seconds. to that in the plasma w27x. In the red cell, the bimolecular reaction involving HU and HbS would be governed by the HbS concentration. Preliminary observations in our lab indicate that the rate of HbS conversion increases by a factor of two when the temperature is increased by 108C. If we take the concentration of HbS in the red blood cell to be 20 mM in heme then Žusing an average bimolecular rate constant for deoxyHbS and oxyHbS at of 1.0 = 10y3 l sy1 .ŽM... we calculate a rate of disappearance of hydroxyurea Ž and concomitant rate of HbS conversion. of 2.0 = 10y5 per second. Assuming this rate would be unaffected by other factors in the cell, it corresponds to 0.13 mM of HbS converted per day. This extremely rough calculation suffers from having ignored other effects on MetHbS formation that would be present in the sickle red blood cell. In the red cell
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the rate of MetHbS formation is complicated by the presence of reducing agents and other oxidizing agents. Our rough calculation of MetHbS formation in vivo is considerably less than what was found in a study where levels of MetHb increased by approximately 0.8 mM Žwith respect to the control. in red cells incubated over 24 h in 0.1 mM hydroxyurea w8x. It is possible that the presence of other oxidants in the red cell could accelerate the reaction between hydroxyurea and hemoglobin. A recent report indicates the participation of hydrogen peroxide Ž found in the sickle red blood cell in elevated levels w28x. in the reaction of hydroxyurea and hemoglobin w19x. Preliminary observations in our laboratories show that the presence of hydrogen peroxide and hydroxyurea results in MetHb formation at a faster rate than the sum of the rates that would be obtained with only one of these reagents present at a time ŽFig. 6.. Although some MetHbS that is formed could be reduced to deoxyHbS in the cell, some of it will take different irreversible pathways and the decrease in oxyHbS and deoxyHbS concentration could be an affect that accumulates over the lifetime of the red cell. An increase in MetHb formation in sickle cell patients would have several negative ramifications. There are many deleterious affects associated with MetHb formation in sickle cell patients Ž for a review
Fig. 6. Time resolved absorption of sickle cell hemoglobin Ž228 mM. and hydroxyurea Ž66 mM. in the presence of hydrogen peroxide Ž2.2 mM.. Seven absorption measurements are shown from 3 ms after mixing to 180 s after mixing, evenly spaced 30 s apart.
see w29x or w30x.. Sickle cell patients have elevated levels of MetHbS due to a higher autooxidation rate of HbS and a lower MetHb reduction rate w29x. The increased levels of MetHb result in increased hemichrome formation, heme loss, and heme associated membranes. These effects together with the increase in oxidants that result from MetHbS formation lead to membrane damage characterized by decreased stability and increased rigidity w28x. A previous study by Ballas et al. studied the rheological properties of red cells from two patients as they underwent hydroxyurea therapy w31x. Both patients responded positively to the treatment with respect to an increase in HbF levels. It was found that the deformability properties of the cells of these patients improved and the membrane stability remained normal. The onset of the improvement of rheological properties associated with treatment with hydroxyurea did not correlate perfectly with an increase in HbF levels w31x. Thus it was suggested that another mechanism may contribute to improvement in red cell rheological properties for patients taking hydroxyurea w31x. In another study, it was found that normal red cells that were incubated in hydroxyurea experienced membrane damage and became more fragile w8x. These results, taken together suggest that the beneficial rheological effects observed by Ballas et al. in vivo resulted from reduced HbS polymerization. The positive effect of MetHbS formation, discussed below, may have played a role. The main positive effect of MetHbS formation for sickle cell patients would be that the tendency for HbS polymerization would decrease as a result of a decreased cellular concentration of deoxyHbS during circulation w30,32x. This would also be the case for HbS converted to the HbNO form. In fact, increasing the level of MetHbS and HbNO have been considered as a treatment for sickle cell disease w30,33x. Even a small change in the concentration of deoxyHbS can have a large effect on whether HbS polymerization occurs because of the effect on the kinetics of polymerization. HbS polymerization is characterized by a long period of time where no polymerization Žknown as the delay time. occurs followed by rapid exponential polymerization w34–37x. Many red blood cells escape HbS polymerization by reaching the venous circulation or being reoxygenated before polymerization begins w38x. The delay time is
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roughly related to the concentration of deoxyHbS by the relation w35x 1 c0 n sl td cs
ž /
where td is the delay time, c 0 is the total HbS concentration, cs is the solubility Žthat is the amount of deoxyHbS that will remain in the solution Žas opposed to the polymer. phase. , is a proportionality factor and n is between 30 and 40. This means that if the concentration of deoxyHbS decreases to 19.7 from 20 mM the delay time Žwith n s 40. becomes 1.8 times longer. Although we have ignored any small changes that would occur involving the solubility of deoxyHbS Žfrom non-ideality considerations. , it is certain that such a change in the cellular concentrations of deoxyHbS due to MetHbS formation would cause large changes in td thus preventing many cells from becoming rigid and contributing to microvascular occlusion. We hypothesize that the beneficial affect that some patients undergoing hydroxyurea therapy experience is due, in part, to MetHbS and possibly HbS-NO formation. Thus MetHbS and HbSNO formation could help to explain improvements in rheological properties w31x and clinical manifestations w4,5x observed in patients taking hydroxyurea. Acknowledgements We thank Yiren Gu for helpful discussion. This work was supported by Wake Forest University startup funds ŽDBK-S., the American Heart Association Žgrant 963031N awarded to SBK. and the National Institutes of Health Comprehensive Sickle Cell Center Grant HL38632 awarded to SKB. References w1x D.L. Rucknagel, in: R.D. Levere ŽEd.., Sickle Cell Anemia and Other Hemoglobinopathies, Academic Press, New York, 1975, p. 1. w2x V.M. Ingram, Nature 178 Ž1956. 792–794. w3x S.H. Embury, R.P. Hebbel, N. Mohandas, M.H. Steinberg, Sickle Cell Disease. Basic Principles and Clinical Practice, Raven Press, New York, 1994. w4x S. Charache, M.L. Terrin, R.D. Moore, G.J. Dover, F.B. Barton, S.V. Eckert, R.P. McMahon, D.R. Bonds, Investigators of the multicenter study of hydroxyurea in sickle cell anemia, New Engl. J. Med. 332 Ž1995. 1317–1322.
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