Amelioration of oxidative stress in red blood cells from patients with [beta]-thalassemia major and intermedia and e-[beta]-thalassemia following administration of a fermented papaya preparation

Phytother. Res. (2010)
Published online in Wiley InterScience
( DOI: 10.1002/ptr.3116
Amelioration of Oxidative Stress in Red Blood
Cells from Patients with β-thalassemia Major

and Intermedia and E-β-thalassemia
Following Administration of a Fermented
Papaya Preparation

Eitan Fibach1*, Ee-Shien Tan2, Saumya Jamuar2, Ivy Ng2, Johnny Amer1
and Eliezer A. Rachmilewitz3
1Department of Hematology, Hadassah – Hebrew University Medical Center, Jerusalem, Israel
2Department of Paediatric Medicine and Genetics Service, KK Women’s and Children’s Hospital, Singapore
3Department of Hematology, The E. Wolfson Medical Center, Holon, Israel
In β-hemoglobinopathies, such as β-thalassemia (thal) and sickle cell anemia, the primary defects are mutations
in the β-globin gene. However, many aspects of the pathophysiology are mediated by oxidative stress. Fer-
mented 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 β-thal patients: β-thal, major and intermedia, (in Israel) and E-β-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 signifi -
cant 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.

Keywords: hemoglobinopathies; thalassemia; erythrocyte; free radicals; antioxidants; fl ow cytometry.
intermedia (in Israel) and β-thal E (in Singapore) with INTRODUCTION
FPP given orally on the oxidative status in their blood cells.
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 (Santiago et al., 1991; Osato et al., 1995; Marcocci et al., 1996; Marotta et al., 1997). In β-hemo- MATERIALS AND METHODS
globinopathies, such as β-thal and sickle cell anemia, the primary defects are mutations in the β-globin gene. FPP. A product of yeast fermentation of Carica papaya
However, many aspects of the pathophysiology are Linn. It was supplied as sachets, each containing 3g mediated by oxidative stress (Hebbel et al., 1982; Rach- powder, by Osato Research Institute, Gifu, Japan. The milewitz and Schrier, 2001; Rund and Rachmilewitz, compositions of its principal components are listed in 2005). RBC ( Amer et al., 2003; 2006) as well as platelets (Amer and Fibach, 2004) and polymorphonuclear leu-kocytes (Amer and Fibach, 2005; Amer et al., 2006) of Patients. The β-thal group included 8 patients with
these patients have increased levels of ROS, membrane β-thal intermedia and 3 with β-thal major (Table 2). lipid peroxidants and exposed PS concomitant with They had different mutations in their β-globin genes. lower levels of GSH compared with their counterparts Those with thalassemia major were frequently trans- fused, while those with thalassemia intermedia were In the present study, we tested the effect of treatment not. Five patients received iron chelation with deferiox- of two groups of thalassemic patients, β-thal major and amine administered subcutaneously for 10 h, 5–7 days a week. In polytransfused patients, blood samples were obtained at least three weeks following the previous * Correspondence to: Eitan Fibach, Department of Hematology, Hadas- transfusion. The E-β-thal group included seven patients. sah – Hebrew University Medical Center, Ein-Kerem, Jerusalem 91120, Israel.
These patients were not on regular blood transfusions and had not received any transfusion 10 weeks prior to Copyright 2010 John Wiley & Sons, Ltd. Table 1. Composition of FPP
The composition analysis was carried out and authenticated by the Japan Food Research Table 2. Patient population
a = deferioxamine treatment; b = intermedia; c = irregularly; d = every 4 weeks.
commencement of the study. Informed consent was Flow cytometry measurements of oxidative stress
obtained in the two groups of patients.
markers. Cells were analyzed either before or after
The β-thal patients were treated with FPP (3 g three 15 min stimulation with 0.5 mM H2O2. For ROS mea- times a day after meals) for three months. The E-β -thal surement, RBC were incubated with 100 μM (fi nal con- group was treated with FPP (3 g two times a day after centration) 2′-7′-dichlorofl uorescin diacetate (DCF) (Sigma, St Louis, MO, USA) for 15 min at 37°C in a Peripheral blood (0.5 ml) samples were obtained humidifi ed atmosphere of 5% CO2 in air. For measuring prior and during treatment. The blood was diluted with GSH content, cell concentrates were incubated for equal volume of Ca++– and Mg++–free Dulbecco’s phos- 3 min at room temperature with [1-(4-chloromercury- phenyl-azo-2-naphthol)] (mercury orange) (Sigma, St Copyright 2010 John Wiley & Sons, Ltd. FPP ANTIOXIDANT EFFECT IN THALASSEMIA PATIENTS Louis, MO, USA) at fi nal concentration of 40 μM, fol- and to phagocytosis by macrophages (Fibach and Rach- lowing washing in PBS. For lipid peroxidation, RBC milewitz, 2008), causing their shorter survival in the suspensions were labeled with 40 μM N- (fl uorescein-5- circulation, and consequently become sensitive to thiocarbamoyl) 1,2-dihexadecanoyl-sn-glycero-3-phos- chronic anemia; neutrophils have reduced capacity to phoethanolamine, triethylammonium salt (Molecular generate oxidative burst – an intracellular mechanism Probes Inc., Eugene, OR,USA) dissolved in ethanol. of bacteriolysis (Wiener, 2003), a possible cause for The cells were incubated for 1 h at 37°C in a humidifi ed recurrent infections; and platelets tend to become acti- atmosphere of 5% CO2 in air, with continuous agitation, vated (Iuliano et al., 1997), leading to frequent throm- centrifuged once to remove unbound label, and re- boembolic complications (Eldor and Rachmilewitz, suspended in PBS. Externalization of PS was deter- 2002). Various mechanisms have been suggested to be mined following washing of the cells in Ca++-binding involved in these defected cellular functions: increased buffer and staining with isothiocyanate-conjugated susceptibility of RBC to hemolysis and phagocytosis is Annexin-V (IQ products, Groningen, the Netherlands) most likely the result of oxidative damage to their mem- brane proteins, band 4.1, band 3 and spectrin (Beneke Following staining, cells were analyzed by fl ow cytom- et al., 2005) and lipids (Shinar and Rachmilewitz, 1990). etry as previously described (Amer et al., 2004). For Neutrophils’ failure to generate oxidative burst may be each parameter the Mean Fluorescence Intensity (MFI) the result of the effect of excess cytosolic ROS on of at least 30,000 RBC was calculated using the FACS- NADPH oxidase activity; possible mechanisms may equipped CellQuestR software (FACS-calibur, Becton- include damage to the cytosolic enzyme components or to the phagosome membrane lipids (Hampton et al., View, CA, USA). In each assay, unstained cells, both 1998). In platelets, oxidants, by increasing ROS, have treated and untreated, served as control. The MFI of been shown to induce their activation [15].
cells stained with DCF, mercury orange and annexin-V We have previously shown that all these functional is proportional to generation of ROS, the content of defects in thal cells could be ameliorated by antioxi- GSH and extent of external PS, respectively; the MFI dants [13], including FPP (Amer et al., 2008). The of fl uor-DHPE-stained cells is reversely proportional to effect of FPP could be due to its high content of glutamic acid, glycine and methionine which are sub-strates for glutathione synthesis (Santiago et al., 1991; Statistical analysis. The results are expressed as the
Osato et al., 1995; Marcocci et al., 1996). In addition, average ± standard deviation (SD) MFI and compared GSH sparing effect could also be an option, since using the two-sample Student’s t-test for differences in decreased generated ROS requires less GSH for their The concept of using phytochemicals such as indicax- anthin (Tesoriere et al., 2006) and curcumin (Thephin- RESULTS AND DISCUSSION
lap et al., 2009) as antioxidants in β thalassemic RBC has shown promising results. FPP was previously dem- Blood samples were drawn and analyzed for ROS and onstrated to have an effect in vivo: oral administration GSH prior and at different times during treatment. into rats showed a signifi cant inhibition of the formation Figure 1 shows the average values for all treated patients of thiobarbituric acid reactive substances, which is an before and during the treatment. In the two groups of index of lipid peroxidation in iron-induced epileptic patients, a marked decrease in ROS and an increase in focus of rats (Santiago et al., 1991), and an increase in GSH were observed in RBC, as well as a decrease in superoxide dismutase activity in their cortex and hip- lipid peroxidation and in externalization of PS residues. pocampus (Imao et al., 1998). In the present study of However, despite the signifi cant changes in all the oxi- oral treatment with FPP of two groups of β-thalassemic dation parameters tested, there were no signifi cant patients with β-thal major and intermedia, and with E- changes in the hematological parameters, including β-thal from different countries showed a signifi cant complete blood count, RBC indices, reticulocytes and reduction in all the tested parameters of oxidative stress in their blood cells, without a signifi cant improvement Oxidative stress in thalassemia is caused primarily by in the hematological parameters. Since the turnover of the RBC abnormalities – degradation of unstable Hb the erythron is relatively slow, it is possible that longer which ends up in free globin chains and heme with duration of treatment is required in order to achieve the eventual iron release. Another contributing factor to latter goals. In addition, a combination treatment with iron overload is increased intestinal absorption and an antioxidant, like FPP, with an iron chelator might regular blood transfusions. Consequently, free iron species – non-transferrin-bound iron (NTBI) and labile plasma iron catalyzing the formation of ROS, have been identifi ed (Esposito et al., 2002).
Chronic oxidative stress in blood cells, such as the This work was partially funded by a grant from Osato Research Insti- case in thalassemia, affects their function: We have pre- tute, Japan. EF and ER are consultants to the Osato Research viously shown that RBC become sensitive to hemolysis Copyright 2010 John Wiley & Sons, Ltd. ROS unstimulated
ROS stimulated
GSH unstimulated
GSH stimulated
LP unstimulated
LP stimulated
PS unstimulated
PS stimulated
Figure 1. Changes in parameters of oxidative stress following administration of FPP to patients with β-thalassemia (__᭿) and E-β
thalassemia (---᭡). The data present the Mean Fluorescence Index (MFI) of cells stained for reactive oxygen species (ROS), reduced
glutathione (GSH), lipid peroxidation (LP) and external phosphatidylserine (PS) in H2O2-stimulated (right) and unstimulated (left)
samples. Note that ROS, GSH and PS for cells stained for LP, the MFI is reversely proportional to their LP. The results show a decrease
in ROS, LP and externalization PS concomitant with an increase in GSH.
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