Thalassemic RBC Research Articles

Antioxidant status in beta thalassemia major: A single-center study

Homozygous β thalassemia may lead to a marked reduction or absence of normal β chain production and accumulation of unpaired alpha-globin chains. A crucial component in the oxidant susceptibility of the thalassemic RBC is the release of heme and iron from the excessive, unpaired α-globin chains. This release can initiate self-amplifying redox reactions, which deplete the cellular reduction potential (e.g., GSH), oxidize additional hemoglobin and accelerate RBC destruction. Furthermore, β-thalassemia patients are under continuous blood transfusion, which, although life-saving, leads to an iron overload with a resultant increase in non-transferrin-bound iron that may cause greater tissue toxicity than iron in other forms. Iron-induced oxidative stress is known to be one of the most important factors determining cell injury in thalassemic patients. Therefore, we designed this study to obtain a comprehensive picture of the iron overload, antioxidant status and cell damage in β thalassemia major patients undergoing regular blood transfusion. A total of 48 diagnosed patients of β thalassemia major and 30 age- and sex-matched healthy subjects were included in the study. Estimation of hemoglobin, hematocrit, glutathione peroxidase (GPX), superoxide dismutase (SOD),vitamin E, serum ferritin, total and direct bilirubin, AST and ALT was carried out. The levels of vitamin E, antioxidant enzymes GPX and SOD were significantly lowered in β thalassemic patients as compared with the control group (P<0.001). Serum total and direct bilirubin, AST and ALT were significantly elevated in thalassemic subjects as compared with the control group, indicating liver cell damage. Thus, our findings indicate that thalassemics are in a state of enhanced oxidative stress and that the administration of selective antioxidants would represent a promising approach toward counteracting oxidative damage and its deleterious effects on the disease status.

Oxidative stress causes membrane phospholipid rearrangement and shedding from RBC membranes—An NMR study

Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the relationship between oxidative stress experienced by RBCs and their phospholipid content and shedding. Using 1H-NMR, we demonstrated a higher lactate/pyruvate ratio, an indicator of oxidative stress, in normal RBCs treated with oxidants ( t-butylhydroxyperoxide and H 2O 2) as well as in β-thalassemic RBCs. Using 31P-NMR, we found 30% more phosphatidylcholine (PC), and unexpectedly, 35% less phosphatidylserine (PS) in the thalassemic RBCs. PS was decreased by treatment with oxidants and increased by anti-oxidants (vitamin C and N-acetyl cysteine); PC showed the opposite behavior. Thalassemic RBCs incubated in phosphate buffered saline produced more PS in the supernatant than normal RBCs. Anti-oxidants reduced the PS in the supernatant while oxidants increased it. Plasma of thalassemic patients contained 2.6-fold and 1.8-fold more PS and PC, respectively, than normal plasma. These results indicate that the decreased PS in RBCs resulted from increased shedding. The nature of the shed PS was studied by purifying and analyzing membranous microparticles from the plasma and RBC supernatants. More PS was found in microparticles purified from thalassemic plasma and RBC supernatants (5.6- and 4.8-fold, respectively) than in their normal counterparts. However, the bulk (80–90%) of the shed PS was not associated with microparticles. The significance of PS shedding for RBC survival needs further clarification.

Oxidative Stress Induces Changes and Shedding of Membrane Phospholipids from Thalassemic RBCs - A NMR Study.

Changes in the membrane phospholipid (PL) asymmetry is one of the hallmarks of apoptosis in nucleated cells. Although mature anucleated RBCs, do not undergo the classical pattern of apoptosis, upon trauma or aging they present changes in the membrane asymmetry. These changes include a flip-flop of phosphatidylserine (PS) from the inner leaflet of the membrane to its outer leaflet. This externalization of PS stimulates RBC phagocytosis by macrophages of the reticulo-endothelial system and their removal from the circulation. Oxidative stress is among the causes of PS externalization on RBCs. In beta-thalassemia and sickle cell disease, although the primary defects are mutations in the globin genes, oxidative stress is thought to mediate part of the damage to the RBCs, and particularly to its membrane, including PS externalization. In the present study, we used Nuclear Magnetic Resonance (NMR) spectroscopy to analyze normal and beta-thalassemic RBCs in order to study the relationship between their oxidative status and the content and shedding of their PL. Using 1H-NMR, we demonstrated a higher lactate/pyruvate ratio in thalassemic RBCs, confirming their state of oxidative stress. Using 31P-NMR, we measured the content of various PLs, and found 30±3% more phosphatidylcholine (PC), and unexpectedly, less PS in thalassemic RBCs than in normal RBCs. The PS was increased in thalassemic RBC, but not in normal RBC, by treatment with anti-oxidants (vitamin C and N-acetyl cysteine) and decreased by oxidants (t-butylhydroxyperoxide and H2O2) in normal and thalassemic RBCs. PC showed the opposite behavior, indicating a correlation between PS and PC contents and the oxidative status. Since RBCs with exposed PS have been reported to be more frequent in thalassemic blood than in normal blood, we hypothesized that the decrease in PS is a result of shedding from the external membrane, either as free PS moieties or as part of membrane vesicles. NMR analysis of blood plasma obtained from normal and thalassemic donors indicated a 2.6-fold and 1.8-fold increase in PS and PC, respectively in the latter plasma. In vitro incubation of RBC produced much higher PS in supernatants derived from thalassemic RBCs compared with those of normal RBCs. Anti-oxidants reduced the PS shedding from thalassemic RBCs into their supernatants while oxidants increased the PS shedding by normal RBCs. RBCs are known to shed membranous particles (termed vesicles or microparticles) in vitro and in vivo during their physiologic and pathological senescence. We studied this point by purifying microparticles from plasma and RBC supernatants of normal and thalassemic donors, and measuring the PLs content in their lipophilic extracts by 31P-NMR. We found that the PS content and its proportion out of the total PLs were higher in microparticles purified from thalassemic plasma (0.25±0.04 mM, 19% of the plasma total PS) or RBC supernatants than in normal plasma microparticles (0.045±0.06 mM, 9.5% of the plasma total PS) or supernatants. The results also show that although microparticles are enriched in PS compared to their intact RBCs, the bulk of the shed PS is not associated with microparticles. These results suggest that oxidative stress in RBCs causes them to shed their PS and that the increase in PC levels maybe be a compensating mechanism. The pathological consequences of these phenomena on the survival of RBCs in thalassemia warrants further study.

Oxidative Stress of RBC in a Murine Model of Beta-Thalassemia Can Be Reversed by Treatment with Antioxidants.

Oxidative stress is a prominent contributor to the premature destruction of RBC as well as anemia in thalassemia and sickle cell anemia. The oxidative status within RBC is maintained by the balance between oxidative systems, such as Reactive Oxygen Species (ROS), and antioxidative systems, such as reduced glutathione (GSH). Using flow cytometric methods, we previously showed that RBC obtained from patients with thalassemia (Amer et al. Eur J Haematol 70:84, 2003; Cytometry 60:73, 2004) or sickle cell anemia (Amer et al. Blood , 104:972a, 2004) exhibit oxidative stress. In the present study, we assessed the extent of RBC oxidative stress and the effects of antioxidant administration, using the thalassemic mouse model Th3/+. This model closely mimics the phenotype observed in patients affected by beta-thalassemia intermedia, such as low hemoglobin levels (7 to 9 gr/dL), splenomegaly and iron overload. In addition, the mature RBC have a shorter life-span and are characterized by anisocytosis, poikilocytosis and hypochromatism.RBC were derived from normal and thalassemic mice before and 4 hours after i.p. injection of the antioxidants N-acetyl cysteine (NAC), vitamin C (Vit. C) or tocotrinol - a mixture of vitamin E derivatives, at a dose of 150 mg/kg. Intracellular ROS was determined in dichlorofluorescin diacetate-stained RBC following stimulation with 2 mM H2O2; GSH content was assessed in RBC stained with mercury orange. Cells were analyzed by flow cytometry: RBC were gated according to size (forward light scatter) and granularity (side light scatter), their fluorescence was measured and the Mean Fluorescence Channel (MFC) was calculated. Fig. 1 shows the average MFC of ROS and GSH of normal and thalassemic mice treated or not treated with anti-oxidants (N=6 in each group).The results show a significantly higher (2.6-fold) production of ROS and lower (three-fold) levels of GSH in RBC from the thalassemic mice versus those in RBC from normal mice. Administration of antioxidants decreased the ROS of normal and thalassemic RBC by 1.4-fold and 2.6-fold, respectively, whereas GSH levels were significantly increased both in the normal (2.7 fold) and in the thalassemic (9.4-fold) RBC. The results show that the RBC of thalassemic mice are under oxidative stress that could be ameliorated by a short antioxidant treatment. Hence, this mouse model recapitulates the oxidative stress found in thalassemic patients and can serve as a model for studying the effects of antioxidant therapy. The flow cytometry methodology used is helpful in following up the results of the treatment and in evaluating its efficacy in reducing oxidative stress. [Display omitted]

The Oxidative Status of Valinomycin-Resistant Normal and Thalassemic Red Cells (RBCs).

Normal high-K+, low-Na+ RBCs, suspended in low-K+ media and permeabilized to K+ with valinomycin, become dehydrated from net loss of KCl and water. A very small fraction of light, normal RBC and larger fractions of light, sickle cell anemia (SCA) and beta-thalassemia RBC were found to be “valinomycin-resistant” (val-res) due to their Na+/K+ gradient dissipation (PNAS 2000; 97: 8050; BLOOD 2000; 96:24b). In thalassemia and SCA, although the primary lesions involve the globin genes, the major damage to the RBC membranes is mediated by oxidative stress. We previously showed (Cytometry 2004; 60:73) that thalassemic RBC have higher reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels than normal RBC before or after in vitro oxidant stress (treatment with hydrogen peroxide). Here, we examined the oxidative status of val-res RBC from normal and beta-thalassemia major blood. RBC suspended in a plasma-like buffer containing 15 mM KCl and 10 mM valinomycin for 45 min were then layered on arabinogalactone (Larex) with density δ=1.091, and spun at 15,000 g for 30 min. Val-res cells were identified as the low density (δ<1.091 g/ml) RBCs recovered from the interphase layer. The percent val-res RBC in beta-thalassemic samples (n = 10), was 84-fold higher (4.2 ± 0.4% (mean ± SD), range 2.5 to 6.0%) than in normal samples (0.05 ± 0.06%, range 0.02 to 0.1%) (n =10). To determine the oxidative status of the RBC, the cells were washed with PBS and stained for intracellular contents of ROS and GSH, using 2′-7′-dichlorofluoresein and Mercury Orange, respectively. RBC were analyzed by flow cytometry, using gating based on size and granularity. The Mean Fluorescence Channel (MFC) for each fluorochrome was computed. The results showed that valinomycin treatment, per se, did not affect ROS and GSH contents: MFC of the stained un-fractionated RBC was similar before and after treatment with valinomycin, indicating that large changes in MCHC had little or no effect on these measurements. In addition, the unfractionated RBC had ROS and GSH values comparable to those of the high density (val-sensitive) RBC which were recovered from the pellet of valinomycin-treated RBC following Larex fractionation. Measurements on six normal and six beta-thalassemic blood samples indicated that in each case val-res RBC had higher ROS (3.5-10 fold) and lower GSH (2.5-8 fold) levels than the unfractionated RBC or the val-sensitive RBC of the same sample. Compared with val-res cells from normal blood, thalassemic val-res RBC had higher capacity to produce ROS (1.7-fold) and had a lower GSH level (1.5-fold) compared with normal val-res RBC. These results confirm that, as with SCA, beta-thalassemia blood contains a higher percent of val-res RBC than normal blood. They show, further, that (i) both normal and thalassemic val-res RBC have higher oxidative status than other cells (val-sensitive) in the same sample; and that (ii) thalassemic val-res RBC have higher oxidative status than val-res RBC in normal blood. The present results are consistent with the possibility that oxidative stress may contribute to the generation of val-res RBCs, but do not establish a cause-effect relationship. Further studies will be needed to elucidate the origin and significance of these cells.

Oxidative Status of Platelets in Normal and Thalassemic Blood.

Thromboembolic complications, possibly involving chronic platelet activation, are an important cause of morbidity and mortality in beta-thalassemia. Oxidative stress, with the generation of reactive oxygen species (ROS), has been suspected to play a role in the pathophysiology of thalassemia and cardiovascular disorders. Previous investigations demonstrated that ROS profoundly affect platelet function and promote platelet activation. Other studies have shown that platelets themselves produce ROS upon activation.In the present study, we adapted flow cytometric techniques to measure oxidative-state markers, ROS generation and reduced glutathione (GSH), using 2′-7′-dichlorofluorescin diacetate and mercury orange, respectively, in platelets. GSH is the major intracellular antioxidant - an important scavenger of ROS. To exclude non-platelets from analysis, a two-parameter (side light scatter and forward light scatter) gate was set. The identity of the gated cells was verified by immunofluorescence staining for CD41 - a platelet-specific antigen. Using these techniques, the average Mean Fluorescence Channel (MFC) values of platelets from 46 normal donors and 22 beta-thalassemic donors were 176 ± 99 vs. 314 ± 81, respectively, for ROS and 319 ± 87 vs. 113 ± 47, respectively, for GSH. These results show that thalassemic platelets contain higher ROS and lower GSH levels than do normal platelets, indicating a state of oxidative stress. The relationship between platelet activation and oxidative status was determined by treating platelets with thrombin (0.1 U/ml), calcium ionophore (5 μM) or phorbol myristate acetate (400 ng/ml). All these treatments caused platelet activation as well as ROS generation; thalassemic platelets were more responsive than platelets from normal controls. In the absence of any known inherent abnormality in thalassemic platelets, the increased oxidative status was attributable to continuous exposure to oxidative insults from extra-platelet sources. Indeed, further investigation indicated that the oxidative status of normal platelets was increased by thalassemic plasma and was inhibited by the iron-chelator Desferoxamin. Iron and hemin, whose levels are increased in thalassemic plasma, stimulated the platelets' oxidative stress. This was also affected by RBC: it was higher in normal platelets incubated with thalassemic RBC than when incubated with normal RBC. Normal RBC stimulated with hydrogen peroxide, a treatment which results in an elevated oxidative status, increased platelet ROS to a greater extent (3.3-fold) than did unstimulated RBC. These results suggest that thalassemic RBC, having higher than normal ROS, mediate oxidative stress in platelets directly, probably by contact or close proximity. Platelet oxidative stress was ameliorated by antioxidants such as N-acetyl-L-cysteine and vitamin C. Treatment with these agents of oxidant-stimulated platelets reduced ROS and enhanced the GSH level.The present results indicate that in thalassemia, platelets are in a state of oxidative stress, causing their chronic activation and possibly thromboembolic consequences. This situation may also prevail in other RBC anomalies, such as sickle cell anemia, Polycythemia Vera and Paroxysmal Nocturnal Hemoglobinuria, which are also associated with thromboembolic phenomena. Our findings raise the possibility of using antioxidants in addition to antithrombotic drugs as prophylactic treatment in these diseases.

Flow cytometric analysis of the oxidative status of normal and thalassemic red blood cells.

The oxidative status of cells has been shown to modulate various cell functions and be involved in physiological and pathological conditions, including hereditary chronic anemias, such as thalassemia. It is maintained by the balance between oxidants, such as reactive oxygen species (ROS), and antioxidants, such as reduced glutathione (GSH). We studied peripheral RBC derived from normal and thalassemic donors. Flow cytometric methods were used to measure (1) generation of ROS; (2) the content of reduced GSH; and (3) peroxidation of membrane lipids as an indication of membrane damage. ROS and lipid peroxidation were found to be higher, and GSH lower, in thalassemic RBC compared with normal RBC, both at baseline as well as following oxidative stress, such as exposure to hydrogen peroxide. To simulate a state of iron overload, normal RBC were exposed to extracellular ferric ammonium citrate or hemin, or their Hb was denatured by phenylhydrazine. All these treatments increased ROS and lipid peroxidation and decreased GSH. These effects were reversed by N-acetyl cysteine, a known ROS scavenger. Flow cytometry can be useful for measuring oxidative stress and its effects on RBC in various diseases and for studying various chemical agents as antioxidants.

Impaired erythrocyte NAD synthesis: a metabolic abnormality in thalassemia.

We have recently demonstrated that phosphoribosylpyrophosphate (PRPP) synthetase activity is decreased in RBC from individuals with thalassemia minor. Because NAD biosynthesis requires PRPP, the product of the PRPP synthetase reaction, we have investigated NAD synthesis in thalassemic RBC. NAD synthesis was measured in intact RBC both by following the accumulation of unlabeled NAD and by following the incorporation of 14C-nicotinic acid into NAD. Using both assay systems, we demonstrate that NAD synthesis is decreased significantly in thalassemic RBC compared to either normal or high reticulocyte red cells. Although this suggested that NAD content should be decreased in thalassemic RBC, no significant difference in NAD content was found among thalassemic, normal, or high reticulocyte red cells. Mechanisms for the lack of a significant decrease in NAD content in thalassemic RBC are discussed. These results indicate that NAD synthesis is impaired in thalassemic RBC possibly as a result of their decrease in PRPP synthetase activity. Our data provide evidence that thalassemic RBC have secondary metabolic abnormalities in addition to their primary defect in hemoglobin synthesis.

Erythrocyte membrane skeleton abnormalities in severe beta-thalassemia

The protein composition of ghosts, inside-out vesicles (IOV), and membrane skeletons (MS) of erythrocytes (RBC) from splenectomized (spx) and nonsplenectomized (non-spx) patients with beta-thalassemia major and beta-thalassemia intermedia was determined. Ghosts from spx thalassemia intermedia patients had a significant increase in their globin content (which was mostly heme reactive) and contained extra polypeptides in the protein 4.2 to 5 and 6-globin areas. The Triton-extracted MS from all of the thalassemic patients showed two major abnormalities: they retained up to twice the amount of protein 3 when compared with controls; they had a significant increase in their globin content, the concentration of which was independent of their protein 3 content. Analysis of the IOV revealed no differences between those prepared from normal controls and those of the patients. MS from spx thalassemia intermedia patients were grossly abnormal when examined by scanning electron microscopy and they exhibited aggregates of material that on transmission electron microscopy suggested the presence of globin precipitates. We propose that, although the integral protein composition, as reflected in the IOV, from severely affected beta-thalassemics is intact, their MS assembly is deranged. The altered skeletal structure of thalassemic RBC could result from attachment of denatured globin to the skeleton components. These abnormalities may contribute to the premature cell death seen in severe beta-thalassemia.

Erythrocyte Membrane Skeleton Abnormalities in Severe β-Thalassemia

AbstractThe protein composition of ghosts, inside-out vesicles (IOV), and membrane skeletons (MS) of erythrocytes (RBC) from splenectomized (spx) and nonsplenectomized (non-spx) patients with β-thalassemia major and β-thalassemia intermedia was determined. Ghosts from spx thalassemia intermedia patients had a significant increase in their globin content (which was mostly heme reactive) and contained extra polypeptides in the protein 4.2 to 5 and 6-globin areas. The Triton-extracted MS from all of the thalassemic patients showed two major abnormalities: (a) they retained up to twice the amount of protein 3 when compared with controls; (b) they had a significant increase in their globin content, the concentration of which was independent of their protein 3 content. Analysis of the IOV revealed no differences between those prepared from normal controls and those of the patients. MS from spx thalassemia intermedia patients were grossly abnormal when examined by scanning electron microscopy and they exhibited aggregates of material that on transmission electron microscopy suggested the presence of globin precipitates. We propose that, although the integral protein composition, as reflected in the IOV, from severely affected β-thalassemics is intact, their MS assembly is deranged. The altered skeletal structure of thalassemic RBC could result from attachment of denatured globin to the skeleton components. These abnormalities may contribute to the premature cell death seen in severe β-thalassemia.© 1987 by Grune & Stratton, Inc. 0006-4971/87/7001-0024$ 3.00/0

Alterations in membrane protein and phosphorylation pattern in beta-thalassemic red blood cells.

The architecture and phosphorylation pattern of RBC membranes were studied in intact RBC and ghosts of patients with beta-thalassemia intermedia. Electron microscopic studies showed severe morphological alterations in ghosts from spx thalassemic patients. The polypeptide pattern obtained on SDS-PAGE revealed a fourfold increase in globin content of ghosts from spx patients. In addition, multiple protein bands were detected migrating below band 4.2, accompanied by alterations in the band 3 zone. When membrane protein phosphorylation was examined by SDS-PAGE and auto-radiography following incubation of intact RBC with [32P]Pi, a reduced labeling of the normally phosphorylated polypeptides was found in the thalassemic RBC. In addition, new phosphorylated peptides appeared in the region of band 3 and below band 4. On the other hand, phosphorylation of isolated membranes with [gamma-32P]ATP showed no major differences in the labeling of the major phosphorylated proteins. An analysis of the initial rate of spectrin-band 2.1 phosphorylation obtained by counting the excised bands from the SDS gels showed that there was a twofold increase in spectrin-band 2.1-phosphorylation rate catalyzed by cAMP-dependent protein-kinase in the thalassemic ghosts, although no differences were found in the extent of spectrin phosphorability. The results are consistent with major changes in membrane protein disposition in thalassemic RBC, most probably caused by the precipitation of excess globin chains.

Impaired Erythrocyte Calcium Homeostasis in β-Thalassemia

Intracellular calcium (Ca) concentration in erythrocytes (RBCs) is controlled by a low passive influx through a relatively impermeable membrane and by active efflux catalyzed by Ca2+,Mg2+-ATPase. Since precipitation of alpha-globin chains in thalassemic RBCs may interfere with normal membrane function, we studied the RBC intracellular Ca content and the RBC membrane Ca2+,Mg2+-ATPase activity in two groups of patients with nonsplenectomized (n = 9) and splenectomized (n = 9) beta-thalassemia intermedia and in two groups of matched controls. The mean +/- SD Ca concentration in the nonsplenectomized (n = 12) and splenectomized (n = 6) controls were 6.1 +/- 6.0 and 5.8 +/- 3.4 mumol Ca per liter of RBCs, respectively, compared with 26.0 +/- 7.6 (P less than .001) and 85 +/- 24.4 (P less than .001) in the nonsplenectomized and splenectomized thalassemia patients, respectively. The mean +/- SD Ca2+,Mg2+-ATPase activity in the eight nonsplenectomized patients was 0.77 +/- 0.58 mumol inorganic phosphate (Pi) per milligram of protein per hour compared with 0.66 +/- 0.41 in the controls (P = NS). Similar values were obtained for the splenectomized patients and their controls. No correlation was found between either the intracellular Ca content or the Ca2+,Mg2+-ATPase activity with the peripheral nucleated RBC count. These findings suggest that there is a major defect in the membrane of the thalassemic RBC leading to an increased Ca content that is more pronounced in splenectomized patients.

Impaired erythrocyte calcium homeostasis in beta-thalassemia

ntracellular calcium (Ca) concentration in erythrocytes (RBCs) is controlled by a low passive influx through a relatively impermeable membrane and by active efflux catalyzed by Ca2+,Mg2+-ATPase. Since precipitation of alpha-globin chains in thalassemic RBCs may interfere with normal membrane function, we studied the RBC intracellular Ca content and the RBC membrane Ca2+,Mg2+-ATPase activity in two groups of patients with nonsplenectomized (n = 9) and splenectomized (n = 9) beta- thalassemia intermedia and in two groups of matched controls. The mean +/- SD Ca concentration in the nonsplenectomized (n = 12) and splenectomized (n = 6) controls were 6.1 +/- 6.0 and 5.8 +/- 3.4 mumol Ca per liter of RBCs, respectively, compared with 26.0 +/- 7.6 (P less than .001) and 85 +/- 24.4 (P less than .001) in the nonsplenectomized and splenectomized thalassemia patients, respectively. The mean +/- SD Ca2+,Mg2+-ATPase activity in the eight nonsplenectomized patients was 0.77 +/- 0.58 mumol inorganic phosphate (Pi) per milligram of protein per hour compared with 0.66 +/- 0.41 in the controls (P = NS). Similar values were obtained for the splenectomized patients and their controls. No correlation was found between either the intracellular Ca content or the Ca2+,Mg2+-ATPase activity with the peripheral nucleated RBC count. These findings suggest that there is a major defect in the membrane of the thalassemic RBC leading to an increased Ca content that is more pronounced in splenectomized patients.

Proteolysis in thalassemia: studies with protease inhibitors.

Intracellular proteolysis was studied in both thalassemic and normal reticulocytes. The main experiment comprised a short incubation of RBC with [3H] leucine, extensive washings, and further incubation in the presence of cold leucine and protein synthesis inhibitors. In the aliquots removed at various time intervals, the TCA-soluble radioactivity increased in contrast with the TCA-precipitable cpm, which decreased with no exception on prolonged incubation. Results were more pronounced when the initial incubation was carried out in the presence of a valine analog and were markedly inhibited when phenanthroline was added during the second phase of the experiment. Cell-free system gave similar results. "globin" containing analog residues were degraded easier than free alpha- and beta-globin chains; free alpha or beta Hb chains are not proteolysed. Thalassemic and normal reticulocytes did not show any significant differences. It is proposed that the thalassemic RBC contain efficient proteolytic mechanisms up to the end of their maturation, but these prove incapable of proteolysing the excess alpha chains completely. Chemical manipulations aiming to make those chains more digestible constitute another approach to the treatment of thalassemia.