24~
lhochmm,t ~,t Bi,ptlvswa ..ltm, i l 3t;~l')t)2) 2L', 2511 , 1992 Elsevier Science Publishers B.V. All rights resewcd (Its2~, 44~t9 ~)2/$1)5.0Ii
BBAD1S 60011
Rapid Report
Primaquine-induced superoxide production by/3-thalassemic red blood cells L e o n i d N. Grinberg a, O d e d Shalev b, A d a Goldfarb ~ and Eliezer A. Rachmilewitz ~ " Department of Hematology, Hadassah Unicersity Hospital, Jerusalem (Israel) and ~' Department of Medicine, Hadassah Unicersity Hospital, Jerusalem (Israel) (Received 20 April 1992)
Key words: Superoxide; Red blood cell; Thalassemia; Primaquine; Oxidative stress
Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, /3-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of/3-thalassemic cells to oxidative stress.
Several lines of evidence suggest that /3-talassemic red blood cells (/3-TRBC) are subjected to oxidative damage. These include excessive accumulation of heme and non-heme iron [1]; a decrease in the number of SH-groups in membrane and cytoskeleton proteins [2]; an abnormal ratio of saturated to unsaturated fatty acids [3] and precipitation of hemichromes consisting of oxidized a- and /3-hemoglobin chains [4,5]. Although reactive oxygen species have been implicated in mediating these changes [6], no studies have directly demonstrated elevated oxidant generation in TRBC. The purpose of this study was to determine the level of superoxide production in T R B C as compared with normal RBC. To quantitate superoxide production we used the SOD-inhibitable cytochrome c reduction method [7,8]. Since only very small amounts of superoxide were spontaneously released from normal RBC, we induced superoxide production with primaquine (PQ), the antimalarial drug known to cause oxidative stress in RBC [9-12]. In this report we demonstrate that, in the presence of PQ, /3-thalassemic erythrocytes are capable of producing excessive amounts of superoxide as compared with normal RBC. PQ (diphosphate salt), ferricytochrome c (type VI), superoxide dismutase (from bovine erythrocytes), were
Correspondence to: L. Grinberg, Hematology Laboratory, Hadassah University Hospital, Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel. Abbreviations: G-G, gelatin-glucose solution; Hct, hematocrit; MCV, mean cell volume; NRBC, nucleated red blood cells; PMA, phorbol 12-myristate 13-acetate; PQ, primaquine; SOD, superoxide dismutase; TRBC, thalassemic red blood cells; WBC, white blood cells.
obtained from Sigma; gelatin ('golden normal' mesh) from Riedel-De Haen, Seelze, Hannover, Germany. All reagents were dissolved in PBS, (NaCI 75 mM, N a 2 H P O 4 / N a H E P O 4 75 mM (pH 7.40)). For WBC separation, a mixture consisting of 2% (w/v) gelatin and 0.1% (w/v) glucose in PBS (G-G solution) was freshly prepared before use. Six splenectomized, non-transfused patients with /3thalassemia intermedia wee studied. Blood sample sfrom patients and healthy controls were drawn, after informed consent, into standard EDTA-treated tubes. To prepare RBC suspensions plasma and platelets were removed following low-spin centrifugation (200 × g, 10 min). The method of cell sedimentation in gelatin [13] was modified to remove WBC from the RBC suspension. Briefly, 0.5 ml of the suspension was mixed with equal volume of G-G solution in 1 ml tuberculin plastic syringe and kept vertical for 30 rain. After cell sedimentation, about two-thirds of the erythromass was withdrawn from the bottom of the syringe and the procedure was repeated a second time with a fresh G-G solution. Following WBC separation, the RBC were washed four times in cold PBS, and then used in cytochrome c assay. Counts of WBC and NRBC were performed from Wright stained smears before and after separation procedure. WBC separation in gelatin yielded a marked decrease in the number of WBC (per number of RBC) in the final suspension (Table I). There was no change in RBC distribution, as reflected by similar MCV values before and after separation for either normal or thalassemic samples (Table I). Microscopic examination of the smears did not reveal morphological alterations in
249 TABLE I
t h r e e - c o m p o n e n t system. T h e final f o r m u l a to e s t i m a t e the c o n c e n t r a t i o n o f r e d u c e d c y t o c h r o m e c ( n m o l / m l ) was:
WBC / RBC and erythrocyte MCV in normal and ~-thalassemic blood before and after separation with gelatin
Removal of WBC from the blood samples was performed by a two-step separation procedure in 2% gelatin column as described in the text. WBC, RBC and MCV values were obtained with a Coulter counter and WBC counts have been corrected for the presence of NRBC in the talassemic samples. The data are reported as the mean _+S.E. for six thalassemic and six normal blood samples. Statistical difference was assessed by paired Student's t-test. Normal Before WBC/103 RBC MCV, fl
cyt. c ( r e d ) = 4 7 . 6 A 5 5 o - 9 . 8 A 5 5 7 - 3 1 . 8 A 5 4 2 w h e r e A550 d e n o t e s a b s o r p t i o n at the p o i n t o f m a x i m a l d i f f e r e n c e b e t w e e n the r e d u c e d a n d t h e oxidized form of c y t o c h r o m e c a n d A557a n d A542d e n o t e a b s o r p t i o n at t h e isosbestic p o i n t s of these two forms. T h e level o f s u p e r o x i d e d e t e c t e d in the s a m p l e s was c o r r e c t e d for the c e l l - i n d e p e n d e n t r e a c t i o n a n d exp r e s s e d as n m o l O 2 / m l s a m p l e p e r h. T h e n , the results w e r e r e c a l c u l a t e d to b e e x p r e s s e d as s u p e r o x i d e p r o d u c t i o n p e r R B C volume, R B C n u m b e r a n d H b content. S p o n t a n e o u s s u p e r o x i d e p r o d u c t i o n in 2 % suspension of n o r m a l R B C o b t a i n e d from 14 s e p a r a t e experim e n t s was n o t a b l y low (0.10 + 0.03 nmol O 2 / m l sample p e r h), b a r e l y e x c e e d i n g the levels d e t e c t e d in cell-free blanks. By contrast, t h e a d d i t i o n o f P Q to n o r m a l R B C led to a 20-fold i n c r e a s e in s u p e r o x i d e p r o d u c t i o n , r e a c h i n g 1.92 _+ 0.32 n m o l O 2 / m l s a m p l e p e r h ( P < 0.0001). T o assess w h e t h e r r e s i d u a l W B C c o u l d c o n t r i b u t e to s u p e r o x i d e p r o d u c t i o n , fractions of R B C - f r e e W B C p r e p a r e d c o n v e n t i o n a l l y [14] w e r e i n c u b a t e d at a conc e n t r a t i o n of 20000 W B C / m l u n d e r the s a m e conditions as the RBC. W e f o u n d that, u n d e r s t i m u l a t i o n with P M A (150 n g / m l ) , t h e s e cells p r o d u c e d 2.08_+ 0.53 n m o l O ] / m l p e r h (five d u p l i c a t e s in t h r e e experiments), c o n s i s t e n t with values p u b l i s h e d e l s e w h e r e [15]. H o w e v e r , in the a b s e n c e o f P M A , we w e r e u n a b l e to d e t e c t any s p o n t a n e o u s o r P Q - i n d u c e d s u p e r o x i d e p r o duction. T h e results i n d i c a t e that s u p e r o x i d e d e t e c t e d in the R B C s u s p e n s i o n s did not o r i g i n a t e from c o n t a m inating W B C . W e then, c o m p a r e d P Q i n d u c e d s u p e r o x i d e p r o d u c tion in T R B C with n o r m a l R B C a n d f o u n d i n c r e a s e d p r o d u c t i o n by the f o r m e r (199 + 38 vs. 106 _+ 13 n m o l O 2 / m l R B C p e r h, respectively, P < 0.009). B e c a u s e o f the i n h e r e n t d i f f e r e n c e in M C V b e t w e e n n o r m a l a n d T R B C we r e c a l c u l a t e d the d a t a in t e r m s of the a c t u a l
Thalassemic After
Before
After
1.54_+0.21 0.08_+0.02 3.53_+1.15 0.07-+0.03 P < 0.001 P < 0.02 90.3 +6.9 90.8 -+7.3 77.1 -+5.2 75.6 _+5.7 P > 0.05 P > 0.05
R B C a n d r e s i d u a l W B C a f t e r gelatin t r e a t m e n t . N o visible hemolysis c o u l d b e d e t e c t e d following t h e s e p a ration p r o c e d u r e . I n c u b a t i o n s w e r e c a r r i e d o u t in siliconized glass t u b e s at 37°C for 60 min. A typical s a m p l e c o n t a i n e d glucose (5.5 mM), c y t o c h r o m e c (0.05 mM), P Q (0.4 m M ) a n d PBS (150 m M ) in a total v o l u m e o f 1 ml, i n c l u d i n g 2% o f R B C ( v / v ) . E a c h s a m p l e was p a i r e d with t h e s a m e o n e b u t c o n t a i n i n g S O D (210 U / m l ) . Blanks c o n t a i n i n g all c o m p o n e n t s e x c e p t for R B C w e r e p r e p a r e d to r e c o r d c e l l - i n d e p e n d e n t c y t o c h r o m e c reduction. A f t e r i n c u b a t i o n s a m p l e s w e r e c e n t r i f u g e d (1500 × g, 12 min) a n d the s u p e r n a t a n t s w e r e a s p i r a t e d a n d a n a l y z e d for c y t o c h r o m e c r e d u c t i o n . C y t o c h r o m e c s p e c t r a w e r e r e c o r d e d f r o m 500 to 600 nm. E a c h s a m p l e was s c a n n e d a g a i n s t its c o n t r o l with S O D to p r o d u c e a signal r e f l e c t i n g s u p e r o x i d e - d c pendent cytochrome c reduction. Variations between d u p l i c a t e s d i d not e x c e e d 10%. T o c o r r e c t for possible c o n t a m i n a t i o n with H b a n d for u n a v o i d a b l e d i f f e r e n c e s in c y t o c h r o m e c c o n c e n t r a t i o n s in the p a i r e d cuvettes, the s p e c t r o p h o t o m e t r i c d a t a w e r e p r o c e s s e d as follows. T h e extinction coefficients for t h e r e d u c e d a n d oxid i z e d forms o f c y t o c h r o m e c as well as for oxyHb w e r e u s e d to derive the s p e c t r o p h o t o m e t r i c e q u a t i o n s for a
TABLE II Primaquine-induced supreoxide production by normal RBC vs. TRBC
RBC were incubated in PBS+glucose at 37°C for 60 min in the presence of primaquine (0.4 mM). Superoxide production was measured by external cytochrome c reduction. The data are presented as superoxide production per RBC volume, RBC number and Hb content. The mean+S.E, are reported for six thalassemic and six normal blood samples tested in four separate experiments. Statistical difference was determined by paired Student's t-test. nmol O~/ml RBC per h
nmol O~-/10 l° RBC per h
nmol O~/gHb per h
Normal
Thai.
Norm~h
Thai.
Normal
Thai.
106 + 13 P < 0.009
198 + 38
91 _+10 P < 0.002
137_+ 18
298 + 34 P < 0.006
585 _+105
250 number of RBC present in the assays. Furthermore, since oxyHb is believed to be the major substratc of spontaneous or PQ induced superoxide, we also calculated superoxide production per Hb content in the assays. The results, summarized in Table II, clearly demonstrate that, regardless of the methods of data presentation, T R B C produce significantly more superoxide than normal RBC. Spontaneous superoxide production by normal and T R B C yielded a signal too low for adequate comparative analysis, albeit consistent with the experience of others [16]. PQ induced superoxide production was reprodubibly 20-times higher than the spontaneous one, thereby providing the basis for comparison between T R B C and normal RBC. Notably, PQ at this concentration (0.4 mM) did not cause any measurable hemolysis or MetHB accumulation. It is unlikely that the excess of superoxide production by T R B C may have resulted from WBC contaminating the assays. The procedure we utilized for cell separation yielded a m a r k e d decrease in the W B C / R B C ratio, while the actual generation of superoxide by residual WBC did not reach any measurable extent. The possible difference in cell age could hardly account for the difference in superoxide production, since we did not find a correlation between superoxide production by individual T R B C samples and the numbers of NRBC reflecting a younger cell population. Moreover, the excessive superoxide production by T R B C cannot be attributed to impaired SOD function, since the activity of this enzyme in T R B C is even higher than in normal RBC [17]. The precise mechanism whereby PQ affects RBC to generate superoxide is as yet unclear. However, it is well documented that oxyHb as well as its subunits are capable of spontaneous or induced oxidation to ferric forms with concomitant release of superoxide [18,19]. Secondly, in an areated cell-free solution, PQ has been shown to oxidize N A D P H [11] a n d / o r oxyHb [12] to produce superoxide. Besides, when incubated with RBC the drug causes excessive H 2 0 2 , O H - and metHb formation [9-11]. Therefore, we suggest that PQ, by reacting directly or indirectly with intracellular oxyHb, promotes both its oxidation and further superoxide generation.
The present data support the hypothesis [6] that, in TRBC, the unstable Hb moiety, excess of ~Y-Hb subunits, as well as the abnormally high cytosolic and membrane bound iron, render these cells highly sensitive to oxidative stress. Exposure to oxidative stress probably results in overproduction of superoxide and other reactive oxygen species such as OH and H&)~. Excessive generation of these species has been described by Hebbel et al. [16] and later by Schactcr [20] in sickle RBC, and may reflect a common pathophysiological pathway in the cellular damage in both hemoglobinopathies. References 1 Bauminger, E.K., Cohen, S.G., Ofer, S. and Rachmilewitz, E.A. (1979) Proc. Natl. Acad. Sci. USA 76, 439-443. 2 Kahane, I. and Rachmilewitz, E.A. (1976) lsr. J. Med. Sci. 12, 1t-15. 3 Rachmilewitz, E.A., Lubin, B.H. and Shohet, S.B. (1976) Blood 47, 495 505. 4 Rachmilewitz, E.A. (1974) Semin. Hematol. 11,441-462. 5 Sinar, E., Shalev, O., Rachmilewitz, E.A. and Schrier, S.L. (1987) Blood 70, 158-164. 6 Shinar, E. and Rachmilewitz, E.A, (1990) Semin. Hematol. 27, 70 82. 7 Babior, B.M., Kipnes, S.A. and Curnutte, J.T. (1973) J. Clin. Invest. 52, 741-744. 8 Lynch, R.E. and Fridovich, I. (1978) J. Biol. Chem. 253, 46974699. 9 Cohen, G. and Hochstein, P. (1964) Biochemistry 3, 895-900. 10 Grinberg, L.N. and Allakhverdiev, A.M. (1981) Med. Parazitol. 2, 54-57. 11 Thornalley, P.J., Stern, A. and Bannister, J.V. (1983) Biochem, Pharmacol. 23, 3571-3575. 12 Summerfield, M. and Tudhope, G.R. (1978) Brit. J. Clin. Pharm. 6, 319-323. 13 Jensen, J.B. (1978) Am. J. Trop. Med. Hyg. 27, 1274-1276. 14 Boyum, A. (1976) Scnad. J. Immunol. 5, 9-15. 15 Vercelloni, G.M., Van Asbeck, B,S. and Jacob, tI.S. (1985) J Clin. Invest. 76, 956-962. 16 Hebbel, R.P., Eaton, J.W., Balasingam, M. and Steinberg, M.tt. (1982) J. Clin. Invest. 70, 1253-1259. 17 Conceni, A., Massei, P., Rotilio, G., Brunori, M. and Rachmilewitz, E.A. (1976) J. Lab. Clin. Med. 87, 1057-1064. 18 Scott, M.D., Rouyer-Fessard, P.H., Lubin, B.H. and Beuzard, Y. (1990) J. Biol. Chem. 265, 17953-17959. 19 Brunori, M., Falcioni, G., Fiorreti, E., Giardina, B. and Rotilio, G. (1975) Eur. J. Biochem. 53, 99-104. 20 Schacter, L.P. (1986) Eur. J. Clin. Invest. 16, 2114-210.
lhochmm,t ~,t Bi,ptlvswa ..ltm, i l 3t;~l')t)2) 2L', 2511 , 1992 Elsevier Science Publishers B.V. All rights resewcd (Its2~, 44~t9 ~)2/$1)5.0Ii
BBAD1S 60011
Rapid Report
Primaquine-induced superoxide production by/3-thalassemic red blood cells L e o n i d N. Grinberg a, O d e d Shalev b, A d a Goldfarb ~ and Eliezer A. Rachmilewitz ~ " Department of Hematology, Hadassah Unicersity Hospital, Jerusalem (Israel) and ~' Department of Medicine, Hadassah Unicersity Hospital, Jerusalem (Israel) (Received 20 April 1992)
Key words: Superoxide; Red blood cell; Thalassemia; Primaquine; Oxidative stress
Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, /3-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of/3-thalassemic cells to oxidative stress.
Several lines of evidence suggest that /3-talassemic red blood cells (/3-TRBC) are subjected to oxidative damage. These include excessive accumulation of heme and non-heme iron [1]; a decrease in the number of SH-groups in membrane and cytoskeleton proteins [2]; an abnormal ratio of saturated to unsaturated fatty acids [3] and precipitation of hemichromes consisting of oxidized a- and /3-hemoglobin chains [4,5]. Although reactive oxygen species have been implicated in mediating these changes [6], no studies have directly demonstrated elevated oxidant generation in TRBC. The purpose of this study was to determine the level of superoxide production in T R B C as compared with normal RBC. To quantitate superoxide production we used the SOD-inhibitable cytochrome c reduction method [7,8]. Since only very small amounts of superoxide were spontaneously released from normal RBC, we induced superoxide production with primaquine (PQ), the antimalarial drug known to cause oxidative stress in RBC [9-12]. In this report we demonstrate that, in the presence of PQ, /3-thalassemic erythrocytes are capable of producing excessive amounts of superoxide as compared with normal RBC. PQ (diphosphate salt), ferricytochrome c (type VI), superoxide dismutase (from bovine erythrocytes), were
Correspondence to: L. Grinberg, Hematology Laboratory, Hadassah University Hospital, Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel. Abbreviations: G-G, gelatin-glucose solution; Hct, hematocrit; MCV, mean cell volume; NRBC, nucleated red blood cells; PMA, phorbol 12-myristate 13-acetate; PQ, primaquine; SOD, superoxide dismutase; TRBC, thalassemic red blood cells; WBC, white blood cells.
obtained from Sigma; gelatin ('golden normal' mesh) from Riedel-De Haen, Seelze, Hannover, Germany. All reagents were dissolved in PBS, (NaCI 75 mM, N a 2 H P O 4 / N a H E P O 4 75 mM (pH 7.40)). For WBC separation, a mixture consisting of 2% (w/v) gelatin and 0.1% (w/v) glucose in PBS (G-G solution) was freshly prepared before use. Six splenectomized, non-transfused patients with /3thalassemia intermedia wee studied. Blood sample sfrom patients and healthy controls were drawn, after informed consent, into standard EDTA-treated tubes. To prepare RBC suspensions plasma and platelets were removed following low-spin centrifugation (200 × g, 10 min). The method of cell sedimentation in gelatin [13] was modified to remove WBC from the RBC suspension. Briefly, 0.5 ml of the suspension was mixed with equal volume of G-G solution in 1 ml tuberculin plastic syringe and kept vertical for 30 rain. After cell sedimentation, about two-thirds of the erythromass was withdrawn from the bottom of the syringe and the procedure was repeated a second time with a fresh G-G solution. Following WBC separation, the RBC were washed four times in cold PBS, and then used in cytochrome c assay. Counts of WBC and NRBC were performed from Wright stained smears before and after separation procedure. WBC separation in gelatin yielded a marked decrease in the number of WBC (per number of RBC) in the final suspension (Table I). There was no change in RBC distribution, as reflected by similar MCV values before and after separation for either normal or thalassemic samples (Table I). Microscopic examination of the smears did not reveal morphological alterations in
249 TABLE I
t h r e e - c o m p o n e n t system. T h e final f o r m u l a to e s t i m a t e the c o n c e n t r a t i o n o f r e d u c e d c y t o c h r o m e c ( n m o l / m l ) was:
WBC / RBC and erythrocyte MCV in normal and ~-thalassemic blood before and after separation with gelatin
Removal of WBC from the blood samples was performed by a two-step separation procedure in 2% gelatin column as described in the text. WBC, RBC and MCV values were obtained with a Coulter counter and WBC counts have been corrected for the presence of NRBC in the talassemic samples. The data are reported as the mean _+S.E. for six thalassemic and six normal blood samples. Statistical difference was assessed by paired Student's t-test. Normal Before WBC/103 RBC MCV, fl
cyt. c ( r e d ) = 4 7 . 6 A 5 5 o - 9 . 8 A 5 5 7 - 3 1 . 8 A 5 4 2 w h e r e A550 d e n o t e s a b s o r p t i o n at the p o i n t o f m a x i m a l d i f f e r e n c e b e t w e e n the r e d u c e d a n d t h e oxidized form of c y t o c h r o m e c a n d A557a n d A542d e n o t e a b s o r p t i o n at t h e isosbestic p o i n t s of these two forms. T h e level o f s u p e r o x i d e d e t e c t e d in the s a m p l e s was c o r r e c t e d for the c e l l - i n d e p e n d e n t r e a c t i o n a n d exp r e s s e d as n m o l O 2 / m l s a m p l e p e r h. T h e n , the results w e r e r e c a l c u l a t e d to b e e x p r e s s e d as s u p e r o x i d e p r o d u c t i o n p e r R B C volume, R B C n u m b e r a n d H b content. S p o n t a n e o u s s u p e r o x i d e p r o d u c t i o n in 2 % suspension of n o r m a l R B C o b t a i n e d from 14 s e p a r a t e experim e n t s was n o t a b l y low (0.10 + 0.03 nmol O 2 / m l sample p e r h), b a r e l y e x c e e d i n g the levels d e t e c t e d in cell-free blanks. By contrast, t h e a d d i t i o n o f P Q to n o r m a l R B C led to a 20-fold i n c r e a s e in s u p e r o x i d e p r o d u c t i o n , r e a c h i n g 1.92 _+ 0.32 n m o l O 2 / m l s a m p l e p e r h ( P < 0.0001). T o assess w h e t h e r r e s i d u a l W B C c o u l d c o n t r i b u t e to s u p e r o x i d e p r o d u c t i o n , fractions of R B C - f r e e W B C p r e p a r e d c o n v e n t i o n a l l y [14] w e r e i n c u b a t e d at a conc e n t r a t i o n of 20000 W B C / m l u n d e r the s a m e conditions as the RBC. W e f o u n d that, u n d e r s t i m u l a t i o n with P M A (150 n g / m l ) , t h e s e cells p r o d u c e d 2.08_+ 0.53 n m o l O ] / m l p e r h (five d u p l i c a t e s in t h r e e experiments), c o n s i s t e n t with values p u b l i s h e d e l s e w h e r e [15]. H o w e v e r , in the a b s e n c e o f P M A , we w e r e u n a b l e to d e t e c t any s p o n t a n e o u s o r P Q - i n d u c e d s u p e r o x i d e p r o duction. T h e results i n d i c a t e that s u p e r o x i d e d e t e c t e d in the R B C s u s p e n s i o n s did not o r i g i n a t e from c o n t a m inating W B C . W e then, c o m p a r e d P Q i n d u c e d s u p e r o x i d e p r o d u c tion in T R B C with n o r m a l R B C a n d f o u n d i n c r e a s e d p r o d u c t i o n by the f o r m e r (199 + 38 vs. 106 _+ 13 n m o l O 2 / m l R B C p e r h, respectively, P < 0.009). B e c a u s e o f the i n h e r e n t d i f f e r e n c e in M C V b e t w e e n n o r m a l a n d T R B C we r e c a l c u l a t e d the d a t a in t e r m s of the a c t u a l
Thalassemic After
Before
After
1.54_+0.21 0.08_+0.02 3.53_+1.15 0.07-+0.03 P < 0.001 P < 0.02 90.3 +6.9 90.8 -+7.3 77.1 -+5.2 75.6 _+5.7 P > 0.05 P > 0.05
R B C a n d r e s i d u a l W B C a f t e r gelatin t r e a t m e n t . N o visible hemolysis c o u l d b e d e t e c t e d following t h e s e p a ration p r o c e d u r e . I n c u b a t i o n s w e r e c a r r i e d o u t in siliconized glass t u b e s at 37°C for 60 min. A typical s a m p l e c o n t a i n e d glucose (5.5 mM), c y t o c h r o m e c (0.05 mM), P Q (0.4 m M ) a n d PBS (150 m M ) in a total v o l u m e o f 1 ml, i n c l u d i n g 2% o f R B C ( v / v ) . E a c h s a m p l e was p a i r e d with t h e s a m e o n e b u t c o n t a i n i n g S O D (210 U / m l ) . Blanks c o n t a i n i n g all c o m p o n e n t s e x c e p t for R B C w e r e p r e p a r e d to r e c o r d c e l l - i n d e p e n d e n t c y t o c h r o m e c reduction. A f t e r i n c u b a t i o n s a m p l e s w e r e c e n t r i f u g e d (1500 × g, 12 min) a n d the s u p e r n a t a n t s w e r e a s p i r a t e d a n d a n a l y z e d for c y t o c h r o m e c r e d u c t i o n . C y t o c h r o m e c s p e c t r a w e r e r e c o r d e d f r o m 500 to 600 nm. E a c h s a m p l e was s c a n n e d a g a i n s t its c o n t r o l with S O D to p r o d u c e a signal r e f l e c t i n g s u p e r o x i d e - d c pendent cytochrome c reduction. Variations between d u p l i c a t e s d i d not e x c e e d 10%. T o c o r r e c t for possible c o n t a m i n a t i o n with H b a n d for u n a v o i d a b l e d i f f e r e n c e s in c y t o c h r o m e c c o n c e n t r a t i o n s in the p a i r e d cuvettes, the s p e c t r o p h o t o m e t r i c d a t a w e r e p r o c e s s e d as follows. T h e extinction coefficients for t h e r e d u c e d a n d oxid i z e d forms o f c y t o c h r o m e c as well as for oxyHb w e r e u s e d to derive the s p e c t r o p h o t o m e t r i c e q u a t i o n s for a
TABLE II Primaquine-induced supreoxide production by normal RBC vs. TRBC
RBC were incubated in PBS+glucose at 37°C for 60 min in the presence of primaquine (0.4 mM). Superoxide production was measured by external cytochrome c reduction. The data are presented as superoxide production per RBC volume, RBC number and Hb content. The mean+S.E, are reported for six thalassemic and six normal blood samples tested in four separate experiments. Statistical difference was determined by paired Student's t-test. nmol O~/ml RBC per h
nmol O~-/10 l° RBC per h
nmol O~/gHb per h
Normal
Thai.
Norm~h
Thai.
Normal
Thai.
106 + 13 P < 0.009
198 + 38
91 _+10 P < 0.002
137_+ 18
298 + 34 P < 0.006
585 _+105
250 number of RBC present in the assays. Furthermore, since oxyHb is believed to be the major substratc of spontaneous or PQ induced superoxide, we also calculated superoxide production per Hb content in the assays. The results, summarized in Table II, clearly demonstrate that, regardless of the methods of data presentation, T R B C produce significantly more superoxide than normal RBC. Spontaneous superoxide production by normal and T R B C yielded a signal too low for adequate comparative analysis, albeit consistent with the experience of others [16]. PQ induced superoxide production was reprodubibly 20-times higher than the spontaneous one, thereby providing the basis for comparison between T R B C and normal RBC. Notably, PQ at this concentration (0.4 mM) did not cause any measurable hemolysis or MetHB accumulation. It is unlikely that the excess of superoxide production by T R B C may have resulted from WBC contaminating the assays. The procedure we utilized for cell separation yielded a m a r k e d decrease in the W B C / R B C ratio, while the actual generation of superoxide by residual WBC did not reach any measurable extent. The possible difference in cell age could hardly account for the difference in superoxide production, since we did not find a correlation between superoxide production by individual T R B C samples and the numbers of NRBC reflecting a younger cell population. Moreover, the excessive superoxide production by T R B C cannot be attributed to impaired SOD function, since the activity of this enzyme in T R B C is even higher than in normal RBC [17]. The precise mechanism whereby PQ affects RBC to generate superoxide is as yet unclear. However, it is well documented that oxyHb as well as its subunits are capable of spontaneous or induced oxidation to ferric forms with concomitant release of superoxide [18,19]. Secondly, in an areated cell-free solution, PQ has been shown to oxidize N A D P H [11] a n d / o r oxyHb [12] to produce superoxide. Besides, when incubated with RBC the drug causes excessive H 2 0 2 , O H - and metHb formation [9-11]. Therefore, we suggest that PQ, by reacting directly or indirectly with intracellular oxyHb, promotes both its oxidation and further superoxide generation.
The present data support the hypothesis [6] that, in TRBC, the unstable Hb moiety, excess of ~Y-Hb subunits, as well as the abnormally high cytosolic and membrane bound iron, render these cells highly sensitive to oxidative stress. Exposure to oxidative stress probably results in overproduction of superoxide and other reactive oxygen species such as OH and H&)~. Excessive generation of these species has been described by Hebbel et al. [16] and later by Schactcr [20] in sickle RBC, and may reflect a common pathophysiological pathway in the cellular damage in both hemoglobinopathies. References 1 Bauminger, E.K., Cohen, S.G., Ofer, S. and Rachmilewitz, E.A. (1979) Proc. Natl. Acad. Sci. USA 76, 439-443. 2 Kahane, I. and Rachmilewitz, E.A. (1976) lsr. J. Med. Sci. 12, 1t-15. 3 Rachmilewitz, E.A., Lubin, B.H. and Shohet, S.B. (1976) Blood 47, 495 505. 4 Rachmilewitz, E.A. (1974) Semin. Hematol. 11,441-462. 5 Sinar, E., Shalev, O., Rachmilewitz, E.A. and Schrier, S.L. (1987) Blood 70, 158-164. 6 Shinar, E. and Rachmilewitz, E.A, (1990) Semin. Hematol. 27, 70 82. 7 Babior, B.M., Kipnes, S.A. and Curnutte, J.T. (1973) J. Clin. Invest. 52, 741-744. 8 Lynch, R.E. and Fridovich, I. (1978) J. Biol. Chem. 253, 46974699. 9 Cohen, G. and Hochstein, P. (1964) Biochemistry 3, 895-900. 10 Grinberg, L.N. and Allakhverdiev, A.M. (1981) Med. Parazitol. 2, 54-57. 11 Thornalley, P.J., Stern, A. and Bannister, J.V. (1983) Biochem, Pharmacol. 23, 3571-3575. 12 Summerfield, M. and Tudhope, G.R. (1978) Brit. J. Clin. Pharm. 6, 319-323. 13 Jensen, J.B. (1978) Am. J. Trop. Med. Hyg. 27, 1274-1276. 14 Boyum, A. (1976) Scnad. J. Immunol. 5, 9-15. 15 Vercelloni, G.M., Van Asbeck, B,S. and Jacob, tI.S. (1985) J Clin. Invest. 76, 956-962. 16 Hebbel, R.P., Eaton, J.W., Balasingam, M. and Steinberg, M.tt. (1982) J. Clin. Invest. 70, 1253-1259. 17 Conceni, A., Massei, P., Rotilio, G., Brunori, M. and Rachmilewitz, E.A. (1976) J. Lab. Clin. Med. 87, 1057-1064. 18 Scott, M.D., Rouyer-Fessard, P.H., Lubin, B.H. and Beuzard, Y. (1990) J. Biol. Chem. 265, 17953-17959. 19 Brunori, M., Falcioni, G., Fiorreti, E., Giardina, B. and Rotilio, G. (1975) Eur. J. Biochem. 53, 99-104. 20 Schacter, L.P. (1986) Eur. J. Clin. Invest. 16, 2114-210.
