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From www.bloodjournal.org by guest on June 17, 2017. For personal use only. + Increased Susceptibility of the Sickle Cell Membrane Ca2+ Mg2+-ATPaseto t-Butylhydroperoxide: Protective Effects of Ascorbate and Desferal By R. Blaine Moore, Todd M. Hulgan, Jerril W. Green, and Lucy D. Jenkins Normal and sickle cell erythrocyte membranes were examined for significant differences in their ATPase activities, thiobarbituric acid reactive products formed (measured relative t o malondialdehyde), membrane protein polymerization, and number of protein-free sulfhydryl groups when treated with 0.5 mmol/L t-butylhydroperoxide (tBHP)for 30 minutes. Isolated sickle cell membranes treated with tBHP produced significantly greater inhibition in both their basal and calmodulin-stimulated Caz+ Mg2+-ATPaseactivities (75% inhibition in both cases) compared with that of control membranes. I n addition, there was significantly more malondialdehyde formed from sickle cell membranes compared with control membranes. Oxidation caused greater protein polymerization in sickle cell membranes compared with normal membranes as demonstrated by the formation of high molecular weight polymers separated on sodium dodecyl sulfate polyacrylamide gels. The number of free sulfhydryl groups present in spectrin and actin decreased more in sickle cell membranes as measured by ’H-N-ethyl maleimide autoradiography and gel scanning. To prevent enzyme inhibition, erythrocyte membranes were treated with tBHP in the presenceof 1mmol/L ascorbate, a potential antioxidant, and 1 mmol/L desferal, an iron chelator. Both ascorbate and desferal added alone with tBHP were effective in preventing inhibition of the basal and calmodulin-stimulated Ca2+ Mg2’-ATPase activities in normal membranes, but in sickle cell membranes only the addition of ascorbate and desferal together offered significant protection. The enhanced oxidation observed with sickle cell membranes can be mimicked in normal white membranes by adding hemoglobin, hemin, or ferrous chloride in the presence of tBHP. In contrast t o hemoglobin, ferrous chloride has the ability t o enhance membrane oxidation in the presence of ascorbate with or without tBHP. Furthermore, the addition of desferal t o these membranes greatly decreased the iron-ascorbatetBHP oxidation of erythrocyte membranes as determined by the sustained ATPase activities and the reduced formation of malondialdehyde. Maximal protection was provided by 1 mmol/L desferal in the presence of 1 mmol/L ascorbate, although some protection was observed even at 10 pmol/L, the lowest concentration tested. These results are discussed in light of the pro- and anti-oxidant effects of ascorbate in the absence and presence of iron and tBHP. 0 1992by The American Society of Hematology. E idants such as vitamin C, vitamin E, and riboflavin are decreased in subjects with sickle cell anemia.6-9 Lipid peroxidation” and reduction in the membrane free sulfhydryl status” have been reported as changes in sickle cells resulting from the proposed oxidative stress. Exposure of erythrocytes or erythrocyte membranes to hydroperoxides, particularly t-butylhydroperoxide (tBHP), can result in a number of membrane changes including lipid peroxidation,I2malondialdehyde f~rmation,”.’~ protein polymeri~ation,’~.’~ and protein amino acid degradation.I6 Enzymes are also inhibited by reactive oxygen species and the Ca2+-ATPaseof erythrocyte membranes is one of the most sensitive of these enzymes.” The Ca2+-ATPaseis an integral membrane protein with a molecular weight of 140,000 daltons.” The enzyme is inhibited by sulfhydryl alkylating agents” and by diamide, a selective sulfhydryl oxidizing agent.” The presence of phospholipids is required for optimal activity of the Ca2+but inhibition of the enzyme by hydroperoxides can occur without significant lipid peroxidation.”.” Hydroperoxide inhibition of the Caz+-ATPaseis enhanced by iron or iron-containing compounds such as hemoglobin and hemin.22 Free iron and iron-containing compounds are believed to act as catalysts in the formation of reactive oxygen species. This is seen in the Fenton reaction, in which hydrogen peroxide and ferrous ion generate a hydroxyl radical: + RYTHROCYTES O F persons with sickle cell anemia are subject to many changes due to the polymerization of hemoglobin S . Some of the membrane changes include: altered ion transport, altered cytoskeletal structure, phosphatidylserine transmembrane movement, and reduced enzyme function.‘ These changes appear to be associated with the formation of dense irreversibly sickled cells, the hallmark of this disease. It has been speculated that the formation of these dense cells may be caused by a transient increase of intracellular calcium. Indeed, alterations in Ca2++ Mg’+-ATPase activities have been shown in sickle cell membranes, particularly that portion stimulated by ~almodulin.2.~ Sickle cell membranes are more oxidatively stressed than normal cells. Sickle cells spontaneously generate more reactive oxygen species including superoxide anion, hydroxyl radical, and hydrogen p e r ~ x i d eAdditionally, .~ antioxFrom the USA Comprehensive Sickle Cell Center, University of South Alabama, Mobile. Submitted May 20, 1991; accepted October 22,1991. Supported by the National Institutes of Health Grant No. P60-HL38639. Presented in part in abstract forms at the 3lst annual meeting of the American Society of Hematology in Boston, MA, 1990, and at the annual meeting of the American Society of Biochemishy and Molecular Biology, New Orleans, LA,1990. Address reprint requests to R. Blaine Moore, PhD, FAIC, USA Comprehensive Sickle Cell Center, University of South Alabama, 2451 Fillingim St, Mobile, A L 3661 7. The publication costs of this article were defiayed in part by page charge payment. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1992 by The American Society of Hematology. 0006-4971I921 7905-0003$3.00/0 1334 + + Fe2++ H 2 0 2+ Fe3+ OH- + .OH Other iron-containing compounds may be able to replace the free iron molecule as the Fenton reagent in this reaction. The membranes of sickle cells possess higher levels of denatured hemoglobins,23hemin, and nonheme iron.Z4,25 It is speculated that the nonheme iron component Blood, Vol79, No 5 (March 1). 1992: pp 1334-1341 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. tBHP EFFECTS OF SICKLE CELL Caz+ + Mg”-ATPase is free iron bound to the membrane, possibly chelated to anionic phospholipids. These iron compounds enhance the oxidative stress in sickle cell membranes, and this has been proposed as the cause for many of the changes that occur in sickle cell anemia, including dehydration and adhesivity.26 Because the Caz+-ATPaseis sensitive to oxidative injury, particularly that catalyzed by the presence of iron or iron-containing compounds, it is proposed that the altered CaZ’-ATPase of sickle cell membranes may be due to iron-induced formation of reactive oxygen species. Consequently, both the sensitivity of sickle cell and normal membranes to treatment with tBHP were examined. Protection against this loss of enzyme activity by ascorbate and desferal was also tested. MATERIALS AND METHODS Preparation of eythrocyte membranes. Blood was obtained with consent from normal and sickle cell donors. The blood was centrifuged at 7,OOOg for 1 minute, and the plasma removed by aspiration. The packed red blood cells (RBCs) were washed four times with 0.9% NaCl solution. The supernatant and bum coat were removed after each washing. White ghosts were made by lysing 2 mL of cells with 30 mL of 10 mmol/L Tris [Tris(hydroxymethyl)-amino methane] buffer (pH 7.4) containing 1 mmol/L EDTA (ethylenediaminetetraaceticacid). Pellets were washed EDTA and twice more with 10 once more with Tris buffer mmol/L Tris buffer (pH 7.4). Membrane pellets were collected after each step by centrifuging the lysed suspension at 15,OOOg for 10 minutes. The procedure for preparing pink ghosts, ie, membranes containing hemoglobin, have been reported previously.” The concentration of hemoglobin in these preparations was measured to be about 2 pmol/L or 4% of the total membrane protein. Membrane protein analysis. Membrane protein was measured routinely by the Bradford procedure*’using bovine plasma gamma globulin as standard. Treatment of membranes with tBHP, ascorbate, and desferal. Suspensions of white ghosts (2 mL, 3 to 5 mg protein/&) were incubated with 0.5 mmol/L tBHP for 30 minutes at 37°C. Ascorbate and/or desferal were present in suspensions at a final concentration of 1 mmol/L where indicated in the figure legends. After incubation, the samples were centrifuged at 15,OOOg for 10 minutes. Two 0.4-mL samples from each supernatant were collected for use in the malondialdehyde measurement. The ghost pellets were washed twice with 15 vol (approximately 10 mL) of 10 mmol/L Tris buffer (pH 7.4). The final ghost pellets were resuspended to 1.0 mL with the same 10 mmol/L Tris pH 7.4 washing buffer. Malondialdehyde (MDA) measurement. MDA was measured by a modification of the procedures described by Bidlack and Tappel.= To each 0.4-mL sample of supernatant or standard was added 0.85 mL of 0.47% 2-thiobarbituric acid and 0.25 mL of 100% trichloroacetic acid. Each tube was placed in a boiling water bath for 15 minutes to develop the color and then cooled in a room-temperature water bath for 5 minutes. Samples were centrifuged at 80% for 15 minutes to sediment the small amount of denatured protein. The absorbance of each sample was measured at 532 nm in a Beckman spectrophotometer (Beckman Instruments Tnc, Fullerton, CA). Standard MDA was prepared by acid hydrolysis of 1,1,3,3-tetramethoxypropane followed by neutralization with NaOH. Levels are expressed in nmoles of malondialdehyde per milligram of protein. It is understood that thiobarbituric acid reactive products other than MDA are formed during membrane oxidation. The results, expressed in nanomoles of MDA per + 1335 milligram of membrane protein, should be considered as thiobarbituric acid-reactive products equivalent to reactive MDA. Total thiobarbituric acid-reactive products associated with the membrane suspension, expressed in nanomoles of MDA per milligram of protein, can be calculated by multiplying the supernatant MDA values by 1.6. In the experiments using ascorbate and/or desferal, appropriate blanks were prepared to compensate for the absorbance contributed by these compounds. ATPase analyses. ATPase activity was measured by a modification of the method of Levine et al,29as described by Moore et al.M In a total assay volume of 0.5 mL the following reagents were used: imidazole buffer, 30 mmol/L, pH 7.0; NaCl, 100 mmol/L; KCI, 20 mmol/L; ouabain, 0.1 mmol/L, MgCl,, 3 mmol/L; EGTA [ethylenebis(oxyethylenenitri1o) tetraacetic acid], 0.1 mmol/L; CaCl,, 0.2 mmol/L; calmodulin, 0.1 mg/mL; ATP, 3 mmol/L. Treated erythrocyte membranes were also added to the suspension. M e ATPase was measured by omitting calcium and calmodulin. Basal Ca2+ Mg2+-ATPasewas measured in the absence of calmodulin. After the reaction was initiated with ATP, samples were incubated at 37°C for 60 minutes. The reaction was terminated by the addition of 0.7 mL ammonium molybdate reagent (0.5% ammonium molybdate, 2% sodium dodecyl sulfate, in 1N sulfuric acid). Color was induced by the addition of 20 pL of ANSA reagent (0.2% l-amino-2-naphthol-4-sulfonic acid, 1.2% sodium sulfite, 1.2% sodium metabisulfite). After 30 minutes, absorbances of the samples were read at 650 nm in a Beckman spectrophotometer. Measurement of nonheme iron. Nonheme iron associated with white erythrocyte membranes was measured using the ferrozine procedure described by Kuross and HebbeLZ 3H-N-ethylmaleimide, sodium dodeql suvate-polyacylamide gel electrophoresis (SDS-PAGE), and autoradiography. Normal and sickle cell membranes (0.15 mL, 3 mglmL), treated with tBHP for various times 0 to 60 minutes (Figs 1 and 2), were incubated with 0.15 mL of ’H-N-ethyl maleimide (1 mmol/L 25 cpm/pmol, ’H-NEM) for 30 minutes at 37T. The membranes were washed twice again with 10 mmol/L HEPES [N-(2-hydroxyethyl)piperazineN’-(2-ethanesulfonic acid)] buffer pY 7.4 and then salubilized for separation by SDS-PAGE. A 10% polyacrylamide gel was used and electrophoresis was performed according to the procedure of Laemmli.” The gel was stained with 0.025% Coomassie brilliant blue in 50% methanol and 10% acetic acid. Destaining was + a - - - - - - - - - --. 0.04 0 10 20 30 40 50 60 Time (min) Fig 1. Effectsof tBHP on normal and sickle erythrocyte membrane Ca2+ Mgz+-ATPaseand calmodulin-stimulatedCaz+ Mgz+-ATPase activities over time. Membranes were treated with 0.5 mmol/L tBHP as described in Mater[als an4 Methods. The values shown are means Normal; (----), with standard deviationsfrom three experiments. (-), sickle 0111; (O), basal Ca2+ Mgz+-ATPase; (O), calmodulin-stimulated CaZ+-ATPase. + + + From www.bloodjournal.org by guest on June 17, 2017. For personal use only. MOORE ET AL 1336 0.0 J 0 10 20 40 Time (min) 30 50 60 I Fig 2. MDA formation in normaland sickle erythrocytemembranes treated with tBHP. Membraneswere treated with 0.5 mmol/ L tBHP as described in Materials and Methods. The values shown are means with standard deviations from three experiments. I-), Normal; (----I, sickle cell; (A),MDA. performed in 30% methanol, 10%acetic acid. The destained gel was then scanned with an ISCO model 1312 scanner (ISCO Inc, Lincoln, NE) and the peaks quantitated using a Spectra Physics SP 4270 integrator (Spectra Physics, San Jose, CA). To prepare the gel for autoradiography, it was placed in “enhance solution” (En‘Hance; NEN Research Products, Boston, MA) for 1 hour followed by water for 30 minutes and then the gel was dried using a slab gel drier (Model 1140; Hoefer Scientific Instruments, San Francisco, CA). For autoradiography,the dried gel was exposed to X A R S Kodak film (Eastman Kodak, Rochester, NY) for 10 days at -75°C. The x-ray film was developed and the autoradiogram peaks scanned and integrated as described above for the gel. ’H-NEM/protein ratios of membrane proteins were calculated by dividing the integrated peak of the autoradiogram band by the integrated peak of the Coomassie-stained gel. RESULTS Effects of tBHP on normal and sickle cell membranes. Incubating a suspension of normal erythrocyte membranes with 0.5 mmol/L tBHP for 60 minutes caused a 50% inhibition of Ca” + Mg”-ATPase activity and a 40% inhibition of calmodulin-stimulated Ca” + Mg”-ATPase, respectively (Fig 1). The same treatment of sickle erythrocyte membrancs caused a basal and a calmodulin-stimulated Ca” + Mg’+-ATPase inhibition of about 75%, respectively (Fig 1). After tBHP treatment for 60 minutes, the formation of MDA in normal membranes increased almost 50%, while in sickle membranes the MDA level increased almost 200% (Fig 2). To assess the formation of high molecular weight protein polymers (HMWP) and protein sulfhydryl status after oxidation, membranes were treated with ’H-NEM and the labeled proteins separated by SDS-PAGE (Fig 3). From the Coomassie-stained gel, it was apparent that a large band (HMWP present at the top of the gel) composed of polymerized membrane proteins, formed during treatment with tBHP. The HMWP band was evident at 10 minutes and became distinct at 30 minutes. This band formed in both normal and sickle cell membranes but it formed more rapidly in the latter. This was most apparent in the NEM-labeled band on the autoradiogram for the 10-minute samples. To dcterminc the loss of free sulfhydryl groups due to oxidation during tBHP incubation, the amount of ’H-NEM per protein band was quantified by integrating the peaks of the autoradiogram and the destained gel and taking the ratio for the corresponding bands. The results showed a 22% decrease in NEM labeling of sickle cell bands 1 + 2 (spectrin) over the 60-minute period and a 45% decrease in NEM labcling of sickle cell band 5 (actin) between 30 and 60 minutes. Although some decrease in label was observed for these bands in normal cells it was less than that seen in sickle cells. The free sulfhydryl groups associated with band 6, glyceraldchyde-3-phosphatedehydrogenase, were oxidized very rapidly on exposure to tBHP. Protein Stain ....... nMwp Untr. , l+ 3* 8 0 Control 10 30 60 mwm rmmmm rqm 3H-NEM Autoradiogram HMWP Untr. 0 1+2 L 3 4.1 4.2 Control 10 30 60 q am.& L.- C - A Sickle Cell 0 10 30 60 B Sickle Cell 0 10 30 60 -=== m Fig 3. SDS-PAGE of isolated normal and sickle cell membranes treated with tBHP (0.5 mmol/L). From left to right: untreated control; normal, 0 minutes; normal, 10 minutes; normal, 30 minutes; normal, 60 minutes; sickle, 0 minutes; sickle, 10 minutes; sickle, 30 minutes; sickle, 60 minutes. HMWP, high molecular weight polymer; numbers on the left refer to the erythrocyte membrane protein bands. (A) Coomassie blue-stained gel; (E) an autoradiogramof the gel shown in (A). From www.bloodjournal.org by guest on June 17, 2017. For personal use only. tBHP EFFECTS OF SICKLE CELL Ca2++ Mg*+-ATPase 1337 Effects of ascorbate and desferal on tBHP inhibition of normal and sickle cell membrane Ca2+ + MgF+-ATPase. Treatment of white erythrocyte membranes with tBHP (0.5 mmol/L) for 30 minutes did not result in significant loss of enzyme activity or formation of MDA. Therefore, to assess the effects of ascorbate and desferal on normal membranes, oxidation was performed on pink ghosts. Treatment of pink erythrocyte membranes from normal subjects with 0.5 mmol/L tBHP caused a significant decrease in both basal and calmodulin-stimulated Caz++ MgZ+-ATPaseactivities (63% and 68%, respectively; Fig 4A). The addition of ascorbate to these membranes prevented the tBHPinduced losses of basal and calmodulin-stimulated activities allowing only 35% and 30% decreases, respectively. Treatment with 1 mmol/L desferal in the presence of tBHP yielded better protection, resulting in 22% and 14% inhibition of the basal and calmodulin-stimulated Caz+-ATPase activities relative to controls. The greatest protection against the loss of activity was obtained when both ascorbate and desferal were added to the membrane suspension containing tBHP. The basal Ca” + M$+-ATPase activity decreased by only 13% of the control, while the calmodulinstimulated Ca2++ M$+-ATPase activity did not decrease at all. In each case the addition of ascorbate, desferal, or ascorbate + desferal resulted in ATPase activities that were not significantly different (Student’s t-test for unpaired variates) relative to those of untreated membranes. The ATPase activities were significantly different from the activities of membranes treated with tBHP alone. The production of MDA increased on treatment with 0.5 mmol/L tBHP. Treatment of the membranes with 1mmol/L ascorbate or 1 mmol/L desferal (both in the presence of tBHP) gave some protection against MDA production. Again, the greatest protection was seen in the samples treated with both ascorbate and desferal, in which only 0.382 nmol MDA/mg membrane protein were produced. Treatment of sickle cell membranes with 0.5 mmol/L tBHP (Fig 4B) resulted in a greater inhibition of basal and calmodulin-stimulated Ca” + MgZ+-ATPaseactivities compared with normal membranes. In contrast to that observed in normal membranes, the addition of 1 mmol/L ascorbate T 2.1 , 1 > - 1.5 w E 1.2 2 0.9 0.6 0.6 a 0.3 0.3 nn 0.0 ~~ Ctl A 1.5 aB y v p 9 W + 4 L E- d > - 1.5 v 0.9 tBHP tBHP tBHP Desf Asc tBHP Asc Desf h 11.8 vi 1.2 f f 1.8 11.8: m .-3 .c T 2.1 h 1.5 0 v , 2.1 and tBHP to sickle cell membranes provided little protection, ie, the ATPase activities of membranes treated with tBHP + ascorbate were not significantly different from the activities from membranes treated with tBHP alone but were still highly significantly different (P < .005) from the ATPase activities of untreated membranes. Likewise, the use of 1 mmol/L desferal was ineffective in significantly reducing the inhibition of the Caz+ + MgZ+-ATPaseby tBHP. However, treatment with both ascorbate and desferal in the presence of tBHP was effective, allowing only 31% and 15% decreases in the basal Ca” + M$+-ATPase and calmodulin-stimulated CaZ++ MgZf-ATPaseactivities, respectively. The levels of MDA production showed significant increases in the samples treated with tBHP and in the samples treated with ascorbate and tBHP. The levels increased from 0.290 nmol/mg in the sickle cell control to 1.147 nmol/mg and 1.225 nmol/mg in the tBHP alone and tBHP + ascorbate samples, respectively. Treatment with both ascorbate and desferal reduced the MDA formation to a greater extent than with desferal alone. Effects of increasing concentrations of desferal on Ca2+ + M$’-ATPase activities in sickle erythrocyte membranes. When a suspension of sickle erythrocyte membranes was treated with tBHP (0.5 mmol/L) and incubated for 30 minutes at 3TC, the basal Caz++ Mg2+-ATPaseactivity was inhibited 71% (Fig 5, left) and the calmodulin-stimulated CaZ++ MgZ+-ATPaseactivity was inhibited 70% (Fig 5, right). Addition of 1 mmol/L ascorbate in the presence of tBHP increased the activities minimally (0.162 pmol/mg/h in the basal, 0.469 pmol/mg/h in the calmodulin-stimulated). Desferal was added in increasing concentrations in the presence of tBHP. In both the basal and the calmodulinstimulated samples, the greatest increase was seen when the membrane suspensions were treated with 1 mmol/L desferal. The basal Caz++ Mgz+-ATPaseactivity increased to 56% of the untreated sample and the calmodulinstimulated CaZ++ MgZ+-ATPaseincreased to 78% of the untreated sample. Effects of ascorbate on tBHP inhibition of normal membranes in the presence of hemoglobin and iron. We have reportedz6 previously that iron or iron-containing com- G gs w 0 1.2 1.2 yE $ .- 0.9 0.9 g 2 0.6 0.6 $ 0.3 0.3 g E v YI > .+ TI C W Q $ 0.0 0.0 Ctl B tBHP tBHP Asc tBHP Desf tBHP Asc Desf + Fig 4. Effects of ascorbate and desferal on Ca2+ Mg2+-ATPaseactivity and MDAformation in pink erythrocytemembranes treated with tBHP. Membraneswere treated with 0.5 mmol/L tBHP, 1 mmol/L ascorbate, and 1 mmol/L desferal as described in Materials and Methods. Samples were incubated for 30 minutes at 37°C. Values shown are means with standard deviations from three experiments. (A) Normal membranes; (6) sickle cell membranes. (0) Basal . Ca‘+ Mg2+-ATPase;(W), calmodulin-stimulated Ca’+ Mg”-ATPase; (B), MDA. + + From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 1338 MOORE ET AL I 0.5 h 2 1 \ 0.4 -0a, E, . h 2 +Untreated $ +Untreated 1.0 \ $ 5 0.8 E, v x ._ > c 2 0.3 ._ T 0.6 a c ..-> c a, 0 a Ld g 0.2 a m + CL 4 +;' 0.4 I c ._ 3 -tBHP 1 4 -tBHP + -0 NLd0.1 0 -2 o.2 0 , 0 I 1 2 3 4 [Desferall (mM) , 5 0 1 2 3 4 [Desferall (mM) pounds, such as hemoglobin and hemin, can enhance the inhibition of erythrocyte membrane Ca2++ Mg*+-ATPase caused by tBHP. Also, ascorbate at a high concentration (20 mmol/L) protects the membrane Caz++ Mgz+-ATPase from oxidative damage by tBHP when hemoglobin or hemin is present. The effective concentrations of ascorbate that protect against tBHP inhibition of ATPase activities and MDA formation in white erythrocyte membranes in the presence of 2 pmol/L hemoglobin are shown in Fig 6. Untreated samples had basal and calmodulin-stimulated Ca2++ M$+ATPase activities of 0.66 and 1.21 Fmol/mg/h, respectively, with minimal MDA formation (0.037 nmol/mg). The addition of 0.5 mmol/L tBHP caused a greater than 90% reduction in ATPase activities and generated 2.36 nmol/mg of MDA. At the lowest concentrations of ascorbate, 10 to i IL Fig 5. Effects of increasing concentrations of desMg'+-ATPase activities in sickle feral on the Ca'+ erythrocyte membranes treated with tBHP and ascorbate. Membranes were incubated at 37°C for 30 minutes with 0.5 mmol/L tBHP, 1mmol/L ascorbate, and increasing concentrations of desferal (0.1 t o 5 mmol/L). (0).Untreated membranes; (A), tBHPtBHP-treated membranes treated membranes; (0). in the presence of ascorbate. 5 100 pmol/L, the major effect observed is a 77% reduction in MDA formed. In contrast, the inhibition of the CaZ++ MgZ+-ATPaseby tBHP was not protected at all by ascorbate over this same concentration range (0 to 100 Fmol/L). Increasing the concentration of ascorbate from 0.1 mmol/L to 0.5 mmol/L provided significant protection for the Ca2++ Me-ATPase with only a 34% reduction in activity, and reduced the MDA formation to near background levels of 0.17 nmol/mg. From these results it was decided that 1 mmol/L ascorbate could be used in experiments as a protective concentration against MDA formation with only a minor loss (30%) of enzyme activity. The effects of ascorbate on tBHP inhibition of ATPase activities and MDA formation in white membranes plus 0.1 mmol/L FeC12-1 mmol/L adenosine diphosphate (ADP) are shown in Fig 7. In contrast to the protective effects of ascorbate with hemoglobin-enhanced tBHP oxidation, ascorinn 1 9 -1.5 0.0 0.2 0.4 0.6 0.8 Ascorbate Concentration (mM) 1 .o Fig 6. Effects of increasing concentrations of ascorbate on tBHPtreated white ghosts in the presence of hemoglobin. Normal white ghosts were incubated for 30 minutes at 37°C in the presence of 0.5 mmol/ L tBHP, 2 pmol/L hemoglobin, and increasing concentrations of ascorbate, 0.05 t o 1 mmol/L. After centrifugation the supernatants were assayed for malondialdehyde (thiobarbituric acid-reactive products). The remaining membrane pellets were washed twice with 10 mmol/L Tris/HCI buffer pH 7.4 and then assayed for ATPase activities. Values shown are the means of duplicate analyses from t w o experiments: (O), basal Ca'+ + MgZ+ATPase; (O), calmodulinstimulated Ca2+ Mg'+-ATPase; (A),MDA. + 2.0 0.2 0.0 0.0 0.2 0.4 0.6 0.8 Ascorbate Concentration (mM) 5 1 .o Fig 7. Effects of increasing concentrations of ascorbate on tBHPtreated white ghosts in the presence of FeCI,-ADP. White membranes were incubated for 30 minutes at 37°C in the presence of 0.5 mmol/L tBHP, 0.1 mmol/L FeCI,, 1 mmol/L ADP, and increasing concentrations of ascorbate, 0.02 t o 1.0 mmol/L. MDA and ATPase procedures were performed as described in Fig 6 and Materials and Methods. Values shown are the means and standard deviations of duplicate analyses from three experiments. (O),Basal Ca2+ Mg'+-ATPase; (0). calmodulin-stimulated Ca" Mg2+-ATPase;(A),MDA. + + From www.bloodjournal.org by guest on June 17, 2017. For personal use only. + Mg2+-ATPase 1339 tBHP EFFECTS OF SICKLE CELL Ca2+ bate increased iron-enhanced tBHP oxidation of white membranes. The basal and calmodulin-stimulated Caz+ + M$+-ATPase activities decreased further and were all but eliminated at ascorbate concentrations of 100 pmol/L. In concert with this loss of enzyme activity, the formation of MDA increased dramatically from 1.2 to 6 nmol/mg membrane protein. At 1 mmol/L ascorbate the MDA formation neared 10 nmol/mg membrane protein, almost an order of magnitude higher than that produced by iron and tBHP alone with membranes. In our previous report,” we showed that iron-enhanced tBHP oxidation caused half-maximal inhibition of Caz+ + M$+-ATPase activities at about 30 pmol/L FeCl,. In Fig 8 we show that ascorbate (1 mmol/L) shifts the effects of iron-tBHP inhibition of enzyme activity and MDA formation to between 5 and 10 p.mol/L FeCI,. The concentration range of FeCI, that produced optimal inhibition of the Caz++ Me-ATPase activities and MDA formation was 20 to 50 pmol/L. This study was repeated with increasing hemoglobin concentrations (50 pmol/L to 2 mmol/L) in the presence of ascorbate and little to no change in ATPase activities or MDA formation was observed (R.B.M., L.D.J.: unpublished results, May 1991). The results of Figs 7 and 8 indicate that low concentrations of ascorbate and iron can cause significant membrane oxidation and loss of enzyme activity. For this reason we measured the endogenous levels of nonheme iron associated with the membranes of normal subjects, subjects with sickle cell anemia, and subjects with other sickling disorders. The results are shown in Table 1. The average membrane nonheme iron level of sickle cell subjects was 11.75 nmol/mg membrane protein, a value significantly higher than that of normal subjects or subjects with sickle cell &thalassemia or SC hemoglobinopathy. These results are similar to those published previously25 for sickle cell subjects, considering the different membrane protein concentrations that would be obtained using gamma globulin rather than albumin as the protein standard in the Bradford >> f 1.2 * -E .- 1.0 0.8 Sp’ thalassemia sc Nonheme Iron (nmolhg) 9 14 3 2 0.07 f 0.12 11.75 2 5.30 0.16 0.13 v 0.2 2.1 1.8 1.8 h 1.5 1.5 1.2 1.2 40 60 [Feci21 80 100 PM Fig 8. Treatment of white erythrocyte membranes with tBHP, ascorbate, and increasing concentrations of FeCI,-ADP. White membranes were treated with 0.5 mmol/L tBHP and 1 mmol/L ascorbate for 30 minutes at 37°C in the presence of increasing concentrationsof FeCI,-ADP. The ratio of FeCI, to ADP was kept constant at 1/10. Values shown are the means of duplicate analyses from three experiments. (O), Basal Caz+ Mg2+-ATPase; (0). calmodulinstimulated Caz++ Mg’+-ATPase; (A), MDA. + 5 -E 0 g : .9 0.3 8 I 0.6 C 0.3 0.0 F 25 0 0.9 P) 20 < ,0001 2.1 5 0.9 ‘5 .5 0.6 0.4 0 PValue assay. Assuming that there are about 6 mg membrane protein per mL of ghost suspension (or per milliliter of packed RBCs), then the average nonheme iron concentration would be about 70 pmol/L. The decrease in iron-ascorbate-tBHP oxidation of erythrocyte membranes by desferal. Because ascorbate was observed to augment the oxidative effects on membranes by tBHP and FeCI,, we performed experiments to determine if chelation of the iron by desferal would alleviate the oxidative injury (Fig 9). As expected, marked reduction in Ca2++ MgZ+-ATPaseactivities and moderate formation of MDA occurred when white membranes were treated with tBHP and FeCI, (100 pmol/L) + ADP (1 mmol/L). The addition of ascorbate to this oxidative system produced a dramatic increase in MDA production from 1.4 to 10.4 nmol/mg protein with complete obliteration of Ca2+ + M$+-ATPase activities. Desferal was found to be very effective in diminishing the oxidative injury caused by FeCl, + tBHP and by FeCI, + tBHP + ascorbate. In the former oxidative system, ie, FeCI, + tBHP, the chelation of iron by desferal might be expected to restore the enzyme activities and MDA formation to those seen with tBHP treatment of white membranes. However, the Ca” + M$+-ATPase activities obtained with the addition of desferal were higher than expected from tBHP alone and this observation was supported by the results for normal white membranes presented in Fig 4A. The addition of desferal to the iron-ascorbate-tBHP oxidative system produced remarkable protection increasing the calmodulin-stimulated activity from 0.036 to 1.033 pmol/mg/h and decreasing the g 0.6 0 % ss aE 2- c g AA N 1T QJ :E * 4 Hemoglobin Type - - c Table 1. Nonheme Iron Levels Associated With Erythrocyte Membranes o.n on Ctl tBHP FetBHP FetBHP FetBHP FetBHP Asc Desf A+D + Fig 9. Effects of tBHP, ascorbate, and desferal on Ca” Mgz+ATPase activities and MDA formation in normal white membranes in the absence and presence of 100 bmol/L FeCI,. White membranes were incubated with 0.5 mmol/L tBHP at 37°C for 30 minutes in the absence or presence of 1 mmol/L ascorbate and/or 1 mmol/L desferal. Values shown are the means and standard deviations of duplicate analyses from four experiments. (0). Basal Ca2+ Mg*+AtPase; (m), calmodulin-stimulated Caz+ Mg”-ATPase; (a), MDA. + + From www.bloodjournal.org by guest on June 17, 2017. For personal use only. MOORE ET AL 1340 MDA production from 10.4 to 0.16 nmol/mg. Although not shown, the presence of ascorbate and iron without tBHP caused about a significant decrease (>50%) in both the basal and calmodulin-stimulated Caz+ Mg2+-ATPase activities that could be prevented by the addition of desferal. + DISCUSSION The effects of tBHP treatment on sickle cell membranes were enhanced relative to those on normal white erythrocyte membranes. In sickle cell membranes this treatment causes significant loss of Caz+ M$'-ATPase activity and is accompanied by an increase in the formation of MDA, a thiobarbituric acid-reactive product of lipid peroxidati~n.'~.'~ Because the loss of enzyme activity was greater and the MDA formation levels were higher in sickle cell membrane samples than in normal membrane samples treated with the same concentration of tBHP (0.5 mmol/L), it became evident that some membrane-associated component of the sickle cell membrane may be acting to catalyze the oxidative effects of tBHP. It has been shown that many iron or iron-containing compounds are found in excess levels in sickle cells,23 and it is also known that some forms of physiologic iron can catalyze the formation of reactive oxygen species through the Fenton reaction. There is even visible evidence of some membrane component present in sickle cells that is not seen in normal cells. After identical washings and lysings (see Materials and Methods), sickle membranes have an amber color, while normal membranes appear white. Based on these findings and observations, one possible cause that was considered was that nonheme iron and/or iron-containing components found in excess levels in the sickle cell membrane catalyze the formation of reactive oxygen species in the presence of tBHP, thereby increasing oxidative damage to the cell. The presence of a membrane-associated iron compound has apparently been demonstrated by the effects of treatment of normal and sickle membranes with 1 mmol/L ascorbate in the presence of tBHP. Ascorbate is known to act as an antioxidant,22and sickle cell patients have been shown to have lower physiologic levels of ascorbic acid and other natural antioxidants such as vitamin E and ribofla~ i n . 6However, .~ it has also been shown that in levels higher + than physiologic (20 kmol/L), ascorbate can react with free iron molecules to catalyze the formation of hydroxyl radic a l ~ . ~ *In- ' ~our experiment, membrane suspensions were treated with 1 mmol/L ascorbate and 0.5 mmol/L tBHP, and incubated for 30 minutes at 37°C. In normal membranes, ascorbate showed protective effects by increasing enzyme activities and decreasing MDA levels relative to samples treated with tBHP alone. In sickle cell membranes, addition of ascorbate gave different results. The enzyme activity was increased only slightly and the MDA levels were also increased over the levels observed in the samples treated with tBHP alone. The ascorbate did not protect against lipid peroxidation as it did in the normal membranes. We propose that this is due to an iron component in the sickle cell membrane that reacts with the ascorbate to form hydroxyl radicals and enhance oxidative injury. If some nonheme iron is bound to the membranes of sickle cells that can react with ascorbate to enhance oxidation, then it is possible that chelation of this iron component with desferal would allow ascorbate to have the same protective effects in sickle cell membranes as it has in normal cell membranes. Indeed, the desferal did have this effect. Furthermore, it seemed reasonable to presume that if a suspension of membranes were treated with both ascorbate and desferal in the presence of tBHP, the desferal would chelate the nonheme iron component, and the ascorbate would be able to act as an anti-oxidant instead of a pro-oxidant. The results support this presumption. On treatment of normal membranes with 1 mmol/L ascorbate and 1 mmol/L desferal in the presence of tBHP, almost total recovery of basal Caz++ Mg2+-ATPaseactivity was observed; a recovery of calmodulin-stimulated ATPase activity to a level higher than the control sample not treated with tBHP was also observed. In sickle cell membranes, treatment with ascorbate and desferal in the presence of tBHP gave significantly higher activities for both the basal and calmodulin-stimulated ATPase and significantly lower levels of MDA production than in the samples treated with ascorbate and tBHP alone. This would support the concept that the use of ascorbate and desferal together would help protect against oxidative damage seen in sickle cell patients, but further investigation must be made to assess any future therapeutic value that these agents may have. REFERENCES 1. Moore RB: Pathophysiologyof the sickle cell, in Mankad VN, Moore RB (eds): Sickle Cell Anemia: Pathophysiology, Diagnosis and Management, New York, NY, Praeger Scientific, 1992 (in press) 2. Dixon E, Winslow RM: The interaction between (Ca2++ Me)-ATPase and the soluble activator (calmodulin) in erythrocytes containing haemoglobin S. Br J Haematol47:391,1981 3. Litosch I, Lee KS: Sickle red cell calcium metabolism: studies on Caz+ + M$'-ATPase and Ca-binding properties of sickle red cell membranes. Am J Hematol8:377,1980 4. Gopinath RM, Vincenzi FF: (Ca2++ Me)-ATPase activity of sickle cell membranes: Decreased activation by red blood cell cytoplasmic activator. Am J Hematol7:303,1979 5. Hebbel RP, Eaton JW, Balasingam M, Steinberg MH: Spontaneous oxygen radical generation by sickle erythrocytes. J Clin Invest 70:1253,1982 6. Jain SK, Williams DM: Reduced levels of plasma ascorbic acid (vitamin C) in sickle cell disease patients: Its possible role in the oxidant damage to sickle cells in vivo. Clin Chim Acta 149:257, 1985 7. Natta C, Stacewicz-Sapuntazakis M, Bhagavan H, Bowen P: Low serum levels of carotenoids in sickle cell anemia. Eur J Haematol41:131,1988 8. Varma RN, Mankad VN, Phelps DD, Jenkins LD, Suskind RM: Depressed erythrocyte glutathione reductase activity in sickle cell disease. Am J Clin Nutr 38:884, 1983 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. tBHP EFFECTS OF SICKLE CELL Ca2+ + MgZ+-ATPase 9. Adelekan DA, Thurnham DI, Adekile AD: Reduced antioxidant capacity in pediatric patients with homozygous sickle cell disease. Eur J Clin Nutr 43:609,1989 10. Jain SK, Shohet SB: A novel phospholipid in irreversibly sickled cells: Evidence for in vivo peroxidative membrane damage in sickle cell disease. Blood 63:362,1984 11. Rank BH, Carlsson J, Hebbel R P Abnormal redox status of membrane-protein thiols in sickle erythrocytes. J Clin Invest 75:1531,1985 12. Jain SK, Shohet S B Calcium potentiates the peroxidation of erythrocyte membrane lipids. Biochim Biophys Acta 64246,1981 13. Jain S K The accumulation of malondialdehyde, a product of fatty acid peroxidation, can disturb aminophospholipid organization in the membrane bilayer of human erythrocytes. J Biol Chem 259:3391,1984 14. Hochstein P, Jain SK: Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging. Fed Proc 40183,1981 15. Moore RB, Brummitt ML, Mankad V N Hydroperoxides selectively inhibit human erythrocyte membrane enzymes. Arch Biochem Biophys 273:527,1989 16. Davies KJ, Goldberg AL: Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes.J Biol Chem 2628220,1987 17. Graf E, Verma AK, Gorski JP, Lopaschuk G, Niggli V, Zurini M, Carafoli E, Penniston JT: Molecular properties of calcium-pumpingATPase from human erythrocytes. Biochemistry 21:4511,1982 18. Hebbel RP, Shalev 0, Foker W, Rank BH: Inhibition of erythrocyte Ca*+-ATPaseby activated oxygen through thiol- and lipid-dependent mechanisms. Biochim Biophys Acta 8628,1986 19. Scutari G, Ballestrin G, Cavaz AL: Diavalent cation dependent ATPase activities of red blood cell membranes: Influence of the oxidation of membrane thiol groups close to each other. J Supramol Struct 14:1,1980 20. Niggli V, Adunyah ES, Carafoli E: Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Caz+-ATPase.J Biol Chem 25623588,1981 21. Snider RL, Moore RB: The role of lipid peroxidation in tert-butylhydroperoxide inhibition of human erythrocyte calcium + magnesium ATPase. Bios 5765,1988 1341 22. Moore RB, Bamberg AD, Wilson LC, Jenkins LD, Mankad V N Ascorbate protects against tert-butyl hydroperoxide inhibition of erythrocyte membrane Ca2+ + Me-ATPase. Arch Biochem Biophys 278:416,1990 23. Asakura T, Minakata K, Adachi K, Russell MO, Schwartz E Denatured hemoglobin in sickled erythrocytes. J Clin Invest 59633,1977 24. Kuross SA, Rank BH, Hebbel RP: Excess heme in sickle erythrocyte inside-out membranes: Possible role in thiol oxidation. Blood 71:876,1988 25. Kuross SA, Hebbel RP: Nonheme iron in sickle erythrocyte membranes: Association with phospholipids and potential role in lipid peroxidation. Blood 72:1278,1988 26. Hebbel RP, Ney PA, Foker W: Autoxidation, dehydration, and adhesivitymay be related abnormalities of sickle erythrocytes. Am J Physio1256:C579,1989 27. Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248,1976 28. Bidlack WR, Tappel AL: Damage to microsomal membrane by lipid peroxidation. Lipids 8:177,1983 29. Levine SN, Berkowitz LR, Orringer EP: Cetiedil inhibition of calmodulin-stimulated enzyme activity. Biochem Pharmacol 33:581,1984 30. Moore RB, Manery JF, Still J, Mankad VN: The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+-ATPase. Biochem Cell Biol 67:137,1989 31. Laemmli UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680,1970 32. Rowley DA, Halliwell B: Formation of hydroxyl radicals from hydrogen peroxide and iron salts by superoxide-and ascorbatedependent mechanisms: Relevance to the pathology of rheumatoid disease. Clin Sci 64649,1983 33. Winterbourn C C Comparison of superoxide with other reducing agents in the biological production of hydroxyl radicals. Biochem J 182:625,1979 34. Miller DM, Aust S D Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation. Arch Biochem Biophys 271:113, 1989 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 1992 79: 1334-1341 Increased susceptibility of the sickle cell membrane Ca2+ + Mg(2+)ATPase to t-butylhydroperoxide: protective effects of ascorbate and desferal RB Moore, TM Hulgan, JW Green and LD Jenkins Updated information and services can be found at: http://www.bloodjournal.org/content/79/5/1334.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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