TIPS- September 1984 371

Erythrocyte auto. oxidation in sickle cell anemia Robert P. Hebbel Deparmeo~t of Medicine, Uniters'to, of Minnesota Medical Sch~d. lk,~ ~ ttmo ,ttemorzal ihaldmg. 420 Delaa~re Street SE, MmneapolL~, MN 55455. t '.%t.

The single.gene disorder, sickle cell anonia affet'.s hundreds of thousands of children in Africa, India, the Middle East and the Medi,erranean region. Occurren :e Ls also high amongst immigrant population.s in Amerk'a and northern Europe. While molecular biologists have recently shown that the anemia can be partially correcttxl br switching on fetal hemoglobin, Robert Hebbei argues a case for therapy based on drugs designed to counter the pathophysiology of the disease, rather than the genetics. The sickle RBC membrane has many abnormalities, including excessive amounts of hemichromes. These denatured ferric hemoglobitts may facilitate the ex(essire auto- oxidation observed in sickle RBC, which generates superoxide and hydro.~-r free radicab. These highly reactive species may then be responsible for a cascade o r inter- related events characterized by dysfunction of the cytoskeleton, lipid bilaver mzd mechanisms for cation homeostasis. Should such speculation prore ralid, it ~ould be rational to aim therapy at the prevention of hemichrome formation or superoxide generation. As in all complex diseases, development of safe and effectire therapeutics is dependent on understanding of tnw pathophysiology.

Sickle cell anemia is best known for the relative insolubility and deoxygenation- induced polymerization of sickle hemo- globin (FIBS). It has long been assumed that this aberrant behavior of HbS i~ entirely responsible for the two basic clinical manifestations of this disease, hemolytic anemia and microvascular occlusion. However, data suggest that the pathophysiology of sickle disease may be profoundly influenced by abnor- malities of the sickle erythrocyte (RBC) membrane. The development of these abnormalities may be related to the instability of HbS and excessive RBC auto-oxidation.

Sickle cell anemia as a membrane disease Investigators have identified many

abnormalities of the sickle RBC mem- brane. Although these generally have been ignored in di~ussions of sickle disease pathophysiology ~, it seems likely that the dysfunctional membrane of sickle RBC plays a fundamental role in disease pathophysiology. For example, each of the factors alleged to be etiological in development of vaso- occlusion may reflect membrane abnor- malities.

The kinetics of reversible deoxygen- ation-induced RBC sickling are such that abnormal slowing of microvascular blood flow may be necessary for sickling of most cells. Such slowing might be induced by: (1) abnormal interactions between sickle RBC and vascular endo- thelium, which may be due to an abnormality of RBC surface charge topography-, (2) permanently and irre- versibly sickled cells (ISC), which are locked into sickled configuration due to undefined abnt, rmalities of cytoskeletal proteins~: or (3) subpopulations of abnormally dense and poorly deform- able RBC, which reflect cellular dehy- dration and increased internal viseosits due to deficient cation homeostasis ~. Likewise, even propensity for HbS poly- merization is related to cellular dehy- dration (and, therefore, to the RBC membrane), since intracellular hemo- globin concentration is the most critical determinant of HbS solubility ~.

Thus, even though development of membrane abnormalities undoubtedly is due to presence of the mutant gene product (HbS), sickle cell anemia is. in a sense, a membrane disease, it follows that an understanding of the patho-

genesis of these membrane almln'mahtic~ may be u.~ful in dc~'elopment of effe¢- live therapeutics.

Amo-oxidali0n and the sickle RIK, memlmme

Several investigato~ have d¢.~ribcd abnormalities which ma) be "hx~tprlnts" of excessive sickle RBC auto-oxidation These include diminished levels ot vitamin E (Ref. 5) and increa.~d amounts of dialdehvde byproduct., of lipid peroxidation ~' ('malondial~hyde') In addition, excessive crosslinking of amira)-phospholipids by malondialdeh~dc has been identified in lipid extracts of sickle RBC membranes", and we recently have found evidence for abnormal oxi- dation of membrane protein thiols ~.

Suggesting that the.~ abnormalities are not simply explained by the known abnormal su~eptibility of sickle RBC to peroxidation, we found that sickle RBC sDontaneouslv generate approximatel.~ t~ice-normal amounts of superoxide (O:-). peroxide (H_~O_.). and h.~dr,)xyl radical ( 'OH) "~. Since oxygen undere(~.~ umvalent reductions, the appearance of excessive H:O.. necessarih requires excessive O:- generation, the precise reason for which has not been identified. Clearly. however, the normal daih turnover of methemoglobin" x~ould be one relentless .source of 0_.- generatior. (Fig. 1).

The exce~ive generation of O t t l,, particularly important, since this species of reduced oxygen reacts avidh with a wide variety of organic molecules Clas- sically, the appearance of "OH is ex- plained by the Haber-Weiss reaction (Equation 1). in reality, this mechanism probably involves a "catalytic" transition metal ion. the valence of which c~cles (i.e. Equation 1 = Equation 2 r, lus Equation 3).

0 : - ÷ H : O : - - ~ ' O H + O t t - + 0 - ~.1) H:O: + Fe:" - -

"OH + OH + Fe 3" '2)

O2-- - Fe ~'--. F,.:" + O: :3)

The sickle RBC is rich in potemiail.~ catalytic kms such as various specie.', of cytoplasmic and membrane-associated hemoglobins, as well as abno.,'mai deposits of iron and perhaps copper. However, our initial attention I:as focused upon hemichromes, a family of denatured ferric hemoglobins found in

372

excessive amounts in sickle RBC (Ref. 10). The reason for excessive hemi- chrome precipitation in sickle RBC is not known, but it may be related to the instability of HbS (Ref. 10).

Iron from some hemichromes can, in fact, facilitate O2-/i-i,,O2-driven "OH generation s, and hcmichrom¢s are tightly bound to the sickle RBC mere- brane t°. Here, "OH generation would be relatively sequestered from the cell's antioxidant mechanisms (most of which are cytoplasmic), and it would occur directly adjacent crucial membrane components. Thus, the abnormal pres- ence of a catalytic metal ion within or adjacent to the membrane might enhance the likelihood of "OH-induced mere- brane damage, even if amounts of ambient O2-/H,O2 were normal, in this regard, it is of interest that "OH generation appears to correlate with the

Hb-Fe 2÷ (deoxyHb) ~

0 . . ~ Dat'ly met-Hb

Hb_Fea+Oi (metHb) N (oxyHb) ~ ] k

o~ (HC)

/ ,ck..

"OH

Fig. I. Proposed mechanism fiTr intme~throcytic generation of superoxide (02-) and hydroxyl rad- ical ~'OH) (top and bottom loops, respeai~ly). Top loop: in normal hemoglobin physiology, oxygen is supposed to combine ret~ersibly deoxyhemoglobin (HbP~+). However, even in nomml RBC some oxyhemglobin (shown here in its ferric-superoxy form, with tm electron shared betwtwn oxygen and iron) auto-oxidizes with for- marion of me~emoglobin (HbFe ~+) and 0,.- (Ref 9). Although orecise reasons for excessive 0 , generation and twmichrome (HC) formation with. in sickle RBC are not known, formation of methemoglobin is the tint step in hemi,:hrome precipitation. Bottom loop: data suggest that some hemichromes can facilitate O:-L'H:O,.driven "OH generation s. This appears to involve HC m a biologic 'Fenton reagent.' with O f serving to reduce HC.Fe ~*, which then tttlcl$ with H:O: to form "OH. Since HC is membrane.hound, this places "OH generation d~rectly ad/acem to crucial membrane constituents.

amount of membrane-bound hemi- chrome", and hemichromes develop within normal RBC as they age. Thus, these concepts regarding sickle RBC may have implications for normal RBC senescence as well.

Therapeutic We have proposed that abnom~alities

of sickle RBC membranes are explained by excessive RBC auto-oxit~ation, which is hypothesized to initiate a cascade of highly-interrelated events characterized by dysfunction of the cytoskeleton, lipid bilayer, and mech- anisms for cation homeostasis tl. This hypothesis is speculative, and its valid- ation must await much additional inves- tigation, Nevertheless, it may be reason- able to entertain the possibility that antioxidant therapy might be useful in sickle disease.

Unfortunately, existing data also suggest that auto-oxidation could induce most of the membrane abnormalities through multiple mechanisms (i.e, lipid peroxidation and/or thiol oxidation and/ or assorted secondary effects thereof) tt. This may predict that antioxidant therapy aimed at the middle of the hypothesized cascade of events would be ineffective, especially since the efficacy of such therapy is unlikely to be absolute, in this regard, vitamin E has been reported to reduce ISC counts, but other inves- tigators have been unable to confirm this. While vitamin E may pre!ect the lipid bilayer from peroxidation, it may be ineffective in preventing oxidation of cytoskeletal protein thiols or pertur- bations of cation homeostasis. Thus, the most rational approach would seem to be to direct antioxidant therapy a t the very top of the presumed auto-oxidation cascade of events (e.g. at prevention of hemichrome formation or O f gener- ation). Alternatively, if there is a single most-critical membrane defect, thera- peutic intervention designed for its pre- vention may prove useful. The most attractive candidate at the present time seems to be cellular dehydration, a result of deficient cation homeostasis, Several investigators have begun to explore approaches by which this mem- brane defe~ might be reversed.

It should be emphasized that different cellular abnormalities may determine hemolytic rate and vaso-occlusion. Hence, it would seem prudent to direct attempted therapy only at well-under- stood events, lest intervention improve

TIPS - September I ¢84

one clinical problem but worsen the other. This problem is illustrated by an experiment of nature. Concurrent (1- thalassemia appears to diminish sickle RBC abnormalities in such a way as to lessen hemolytic rate and raise hemo- globin concentration. Yet these patients may actually be somewhat worse off in terrrts of vaso-occlusion. Presumably. this reflects the fact that anemia itself is a major compenmtogy mechanism for the markedly abnormal theology of sickle blood. Thus, what benefits the RBC may be harmful to the patient as a whole. Obviously, sickle cell anemia is an exceedingly complex disease, and it is insufficiently understood. Develop- ment of predictable, safe, and effective therapeutics will require an understand- ing of the true pathophysiology of this disease.

AdmowledMment The author's laboratory is supported

by the National Institutes of Health (HL26139, HL30160, and a Research Career Development Award).

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Jacob, H. S., White J. G, and Eaton, J. W. (1980) J. Clin. Insist. 65. 154-I(/)

3 Lux, S, E., John, K. M. and Kamovsky, M. J. (1976) J. Clin, Im~t. 58. 99.5--963

4 Clark, M. R,, Momson, C. E. and Shobet, S. B, (1978) J. Clin, Incest. 62, 329-337

5 Chiu, D., Vichimky, E,, Yee, M., Kleman, K., Lubin, B. 0982) Ann. NY Amd. Sci. 393, 323--333

6 Dis, S, K. and Nair, R. C, (1980) Br. J. Haemmol, 44, 87-92

7 Rank, B., Hebbel, R. P., Jacob, H, S. and Cadsson, J. (tgs3) Blood 62 (Supl~.) ¢~a (abstr.)

8 Hebbel, R, P., Eaton, J. W., Balasingam, M. and Steinberg, M. H. (1982) J. C/in. Infest, 70, 1253-1259

9 Canell, R, W,, Winterboum, C.C. and Rachndlewilz, E, A. (1975) Brit. J. Haematol. 3O, 259..2M

10 Asakura, T,, Minakata, K., Adacld, K,, Russell, M. O, Schwartz, E, (1977)J. Cfin. Invest. 59, 633-640

I I Hebl~l, R. P. Clin, Haenmtol, (in press)

Robert Hebb¢l received his BA ~ Obcdin College, Ohio, in 1969, and his MD ~om the Univenity of Minneum in 1973, Following a Fellowship in Hematology and Oncology, he became Assimnl PVofea~ of Medicine m the University of Minnesota (1979.-1982) where he is cwren~ As,~,ue Profmor of Medicine.