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
PHYTOTHERAPY RESEARCH
Phytother. Res. (2010)
Published online in Wiley InterScience
(www.interscience.wiley.com)
DOI: 10.1002/ptr.3116
FULL PAPER
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).
Acknowledgements
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.
Copyright 2010 John Wiley & Sons, Ltd.
FPP ANTIOXIDANT EFFECT IN THALASSEMIA PATIENTS
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