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In the present study, we questioned the role of oxidative stress in hereditary spherocytosis (HS), where red blood cells (RBC) have a shortened survival due to primary deficiency in membrane proteins. Using flow cytometry techniques, we showed that RBC derived from 17 HS patients of seven families generate more reactive oxygen species, membrane lipid peroxides, and less reduced glutathione than normal RBC. Following in vitro incubation of HS-RBC from seven patients with a fermentation bioproduct of Carica papaya (fermented papaya preparation (FPP)) with known antioxidative properties, oxidative stress markers were significantly reduced. Similar results were obtained following treatment with FPP for 3 months of 10 adult HS patients, as well as decreased tendency to undergo hemolysis. The hemoglobin levels increased by >1 g/dl, mean corpuscular hemoglobin concentration decreased by >1 g/dl, and the reticulocyte count decreased by 0.93%. Concomitantly, lactic dehydrogenase decreased by 17% and indirect bilirubin by 50%. A significant decrease in malonyldialdehyde was also detected. These data indicate that oxidative stress plays an important role in the pathophysiology of HS which can be ameliorated by an antioxidant such as FPP. Additional clinical trials with FPP and other antioxidants are warranted.

In beta-hemoglobinopathies, such as beta-thalassemia (thal) and sickle cell anemia, the primary defects are mutations in the beta-globin gene. However, many aspects of the pathophysiology are mediated by oxidative stress. Fermented papaya preparation (FPP), a natural health food product obtained by biofermentation of carica papaya, has been shown to limit oxidative stress both in vitro and in vivo. We studied the effect of FPP on two groups of beta-thal patients: beta-thal, major and intermedia, (in Israel) and E-beta-thal (in Singapore). The results indicated that in both groups FPP treatment increased the content of reduced glutathione (GSH) in red blood cells (RBC), and decreased their reactive oxygen species (ROS) generation, membrane lipid peroxidation, and externalization of phosphatidylserine (PS), indicating amelioration of their oxidative status, without a significant change in the hematological parameters. Since the turnover of the erythron is relatively slow, it is possible that longer duration of treatment, probably with the addition of an iron chelator, is required in order to achieve the latter goals., (Copyright 2010 John Wiley & Sons, Ltd.)

On the basis of all the presented data, one can conclude that oxidative stress plays a major role in the pathophysiology of thalassemia and other congenital and acquired hemolytic anemias. Free extracellular (labile plasma iron, LPI) and intracellular (labile iron pool, LIP) iron species that have been identified in thalassemic blood cells are responsible for generation of oxidative stress by catalyzing formation of oxygen radicals over the antioxidant capacity of the cell. Consequently, there is a rationale for iron chelation to eliminate the free-iron species, which in this respect, act like antioxidants. In addition, antioxidants such as vitamin E and polyphenols are also capable of ameliorating increased oxidative stress parameters and, given together with iron chelators, may provide a substantial improvement in the pathophysiology of hemolytic anemias and particularly in thalassemia.

Objectives: To evaluate the hematological profile, oxidative stress, and antioxidant parameters in beta-thalassemia/Hb E patients treated with curcuminoids for 12 months., Design and Methods: Twenty-one beta-thalassemia/Hb E patients were given 2 capsules of 250 mg each of curcuminoids (a total of 500 mg) daily for 12 months. Blood was collected every 2 months during treatment and 3 months after withdrawal and was determined for complete blood count, malonyldialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) in red blood cells (RBC), and non-transferrin bound iron (NTBI) in serum., Results: The increased oxidative stress in beta-thalassemia/Hb E patients was shown by higher levels of MDA, SOD, GSH-Px in RBC, serum NTBI, and lower level of RBC GSH. Curcuminoids administration resulted in improvement of all the measured parameters as long as they were administered. After 3 months withdrawal of treatment, all parameters returned close to baseline levels., Conclusion: Curcuminoids may be used to ameliorate oxidative damage in patients with beta-thalassemia/Hb E disease., (Copyright 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Sickle cell disease (SCD) is basically a red blood cell (RBC) disorder characterised by sickling and haemolysis, but platelets and polymorphonuclear neutrophils (PMN) are also involved. Oxidative damage may play a role in the pathogenesis of SCD. Using flow cytometry, we measured oxidative-state markers simultaneously in RBC, platelets and PMN obtained from 25 normal donors, nine homozygous (SS) patients and six SS/beta-thalassaemia patients. Reactive oxygen species (ROS) and reduced glutathione (GSH) were measured following staining of blood samples with fluorescence probes and gating on specific subpopulations based on size and granularity. Ten- to 30-fold higher ROS production and 20-50% lower GSH content were found in RBC, platelets and PMN from SCD patients versus those of their normal counterparts. This could in part account for the clinical manifestations, such as haemolysis, a hypercoagulable state, recurrent bacterial infections and vaso-occlusive incidences, in SCD. We further showed that exposure of SCD samples to antioxidants, such as N-acetyl-cysteine, vitamin C and vitamin E, decreased their oxidative stress. These results suggest that antioxidant treatment of patients with SCD could reduce oxidative damage to RBC, PMN and platelets, thereby alleviating symptoms associated with their pathology. The flow cytometry techniques presented herein could assist in monitoring the efficacy of such treatment.

The pathophysiology of thalassemia is, to a certain extent, associated with the generation of labile iron in the pathological red blood cell (RBC). The appearance of such forms of iron at the inner and outer cell surfaces exposes the cell to conditions whereby the labile metal promotes the formation of reactive oxygen species (ROS) leading to cumulative cell damage. Another source of iron accumulation results from increased absorption due to decreased expression of hepcidin. The presence of labile plasma iron (LPI) was carried out using fluorescent probes in the FACS. RNA expression of hepcidin was measured in two models of thalassemic mice. Hepcidin expression was also measured in human hepatoma HepG2 cells following incubation with thalassemic sera. LPI was identified and could be quantitatively measured and correlated with other parameters of iron overload. Hepcidin expression was downregulated in the livers of thalassemic mice, in major more than in intermedia. Thalassemic sera down regulated hepcidin expression in HepG2 liver cells. A possible way to decrease iron absorption could be by modulating hepcidin expression pharmacologically, by gene therapy or by its administration. Treatment with combination of antioxidants such as N-acetylcysteine for proteins and vitamin E for lipids in addition to iron chelators could neutralize the deleterious effects of ROS and monitored by quantitation of LPI.

Abstract: Fermented papaya preparation (FPP) (a product of yeast fermentation of Carica papaya Linn) is a food supplement. Studies in chronic and degenerative disease conditions (such as thalassemia, cirrhosis, diabetes and aging) and performance sports show that FPP favorably modulates immunological, hematological, inflammatory, vascular and oxidative stress damage parameters. Neuroprotective potential evaluated in an Alzheimer''s disease cell model showed that the toxicity of the β-amyloid can be significantly modulated by FPP. Oxidative stress trigger apoptotic pathways such as the c-jun N-terminal kinase (JNK) and p38-mitogen activated protein kinase (MAPK) are preferentially activated by pro-inflammatory cytokines and oxidative stress resulting in cell differentiation and apoptosis. FPP modulated the H2O2-induced ERK, Akt and p38 activation with the reduction of p38 phosphorylation induced by H2O2. FPP reduces the extent of the H2O2-induced DNA damage, an outcome corroborated by similar effects obtained in the benzo[a]pyrene treated cells. No genotoxic effect was observed in experiments with FPP exposed to HepG2 cells nor was FPP toxic to the PC12 cells. Oxidative stress-induced cell damage and inflammation are implicated in a variety of cancers, diabetes, arthritis, cardiovascular dysfunctions, neurodegenerative disorders (such as stroke, Alzheimer''s disease, and Parkinson''s disease), exercise physiology (including performance sports) and aging. These conditions could potentially benefit from functional nutraceutical/food supplements (as illustrated here with fermented papaya preparation) exhibiting anti-inflammatory, antioxidant, immunostimulatory (at the level of the mucus membrane) and induction of antioxidant enzymes. [Copyright &y& Elsevier]