G6PD

1. G6PD

2. Antioxidant deficit and enhanced susceptibility to
oxidative damage in individuals with different forms of
a-thalassaemia
Thalassaemia refers to a heterogeneous group of inherited
anaemias characterized by defects in the synthesis of a- and b-
globins (Schrier, 1994; Weatherall, 1998). The genetic defects
of a-thalassaemias are commonly caused by deletion of one or
more of the a-globin genes on chromosome 16. Depending on
the number and position of the deletion(s), a-thalassaemias
can be divided into several subtypes: a-thalassaemia trait, with
two defective loci; haemoglobin H disease, with three
dysfunctional loci; and the most severe form, fetal hydrops
syndrome with Hb Barts, in which all four loci are defective
(Schwartz & Benz, 1991). a-Thalassaemia with two a-globin
gene deletions can be further divided into two major groups.
One group has deletions of one a-globin gene from each
chromosome (i.e. homozygosity or compound heterozygosity
for a+
-thalassaemia deletions); whereas the other group has
deletions of both a-globin genes from the same chromosome
(i.e. ao
-thalassaemia deletion). In Taiwan, the latter group
commonly results from Southeast Asian (SEA) deletion (Ko
et al, 1992). The clinical presentations of different a-thalass-
aemic subtypes vary from mild to moderately severe haemo-
lytic anaemia (Weatherall, 1999). Thalassaemia is rather
common in the Taiwanese population. It has been estima-
ted that around 6–8% of Taiwanese are carriers of the
a-thalassaemic gene (Lin et al, 1991; Hsiao, 1992; Ko et al,
1992). The high incidence of this genetic disorder raises a
concern about the health of affected individuals.
Reactive oxygen species (ROS) are implicated in the
pathogenesis of cancer, immune dysfunction and many
degenerative diseases, such as cardiovascular diseases, cata-
racts, and Alzheimer’s disease (Ruberg et al, 1998; Suematsu &
Tsuchiya, 1998; Blankenberg et al, 2003; Brennan et al, 2003).
There is an intimate relationship between oxidative stress and
anaemia. ROS have been shown to damage and change
membrane properties of erythrocytes. Increased membrane
Mei-ling Cheng,1,2,
* Hung-yao Ho,1,
*
Hsiu-chuan Tseng,1
Chien-Hong Lee,1,2
Lee-yung Shih3
and Daniel Tsun-yee
Chiu1
1
Graduate Institute of Medical Biotechnology &
Department of Medical Biotechnology and
Laboratory Science, Chang Gung University,
Kwei-san, Tao-yuan, Taiwan, 2
Molecular
Diagnosis Laboratory, Department of Clinical
Pathology, Chang Gung Memorial Hospital,
Kwei-san, Tao-yuan, Taiwan, and 3
Department
of Haematology & Oncology, Chang Gung
Memorial Hospital, Kwei-san, Tao-yuan, Taiwan
*These authors contributed equally to this
paper.
Received 12 August 2004; accepted for
publication 4 October 2004
Correspondence: Dr Daniel Tsun-Yee Chiu,
Professor, Graduate Institute of Medical
Biotechnology and Department of Medical
Biotechnology and Laboratory Science, Chang
Gung University, 259 Wen-Hua 1st Road,
Kwei-san, Tao-yuan, Taiwan.
E-mail: dtychiu@mail.cgu.edu.tw
Sum m ar
y
a-Thalassaemia is a common red cell disorder in Taiwan, affecting 6–8% of
Taiwanese. Previous studies have shown that reactive oxygen species are
generated in increased amounts in thalassaemic red cells. This implies the
possible alteration of redox status in thalassaemic patients, which may
adversely affect their health. In the present study, the redox status of patients
with a-thalassaemia trait and haemoglobin H (Hb H) disease was
investigated. Lipid peroxidation, as measured by the level of plasma
thiobarbituric acid reactive substances (TBARS), was increased in
a-thalassaemic patients, with the highest level of TBARS in Hb H disease
patient. The plasma levels of vitamin A, C, and E were significantly lower in
a-thalassaemic patients than in controls. The overall antioxidant capacity in
plasma was inversely correlated with the severity of a-globin gene defect: the
more severe the form of a-thalassaemia, the lower the overall antioxidant
capacity in plasma. Erythrocytes isolated from a-thalassaemia patients had
lower levels of vitamin E, glutathione, catalase and superoxide dismutase. In
addition, these a-thalassaemic red cells were more susceptible to hydrogen
peroxide-induced lipid peroxidation and decrease in deformability. All these
data suggest that the a-thalassaemic patients suffer from increased oxidative
stress and antioxidant deficit, which may complicate the pathophysiology of
a-thalassaemia.
Keywords: a-thalassaemia, lipid peroxidation, oxidative stress, reactive
oxygen species, erythrocytes.
research paper

3. rigidity, decreased deformability, and haemolysis are conse-
quences of oxidative damage to erythrocytes (Lubin & Chiu,
1982; Snyder et al, 1985; Chiu et al, 1989; Cheng et al, 1999a).
Moreover, oxidative insult may result in immune recognition
and eventual removal of red blood cells from the circulation
(Low et al, 1985).
Several lines of evidence suggest that ROS are involved in the
pathogenesis of thalassaemias. It has been shown that ROS are
generated in increased amounts in thalassaemic erythrocytes
because of the presence of excess unmatched globin chains, and
deposition of iron, non-haem iron and haemichromes (Shinar
& Rachmilewitz, 1990; Hebbel, 1991; Schrier & Mohandas,
1992; Anastassopoulou et al, 2000; Schrier, 2002). Continuous
ROS production in thalassaemic individuals may alter their
overall redox status and cause other health problems. This
appears to be the case in b-thalassaemia. Reduction in the
levels of vitamin C, vitamin E, and carotenoids has been
reported in b-thalassaemic patients, especially in those receiv-
ing transfusion therapy (Giardini et al, 1981, 1985; Miniero
et al, 1982; Livrea et al, 1996, 1998; Tesoriere et al, 1998). Low-
density lipoprotein (LDL) isolated from b-thalassaemic
patients has high levels of oxidized lipid and protein compo-
nents (Livrea et al, 1998), and is highly susceptible to oxidative
modification (Tesoriere et al, 1998). The latter may account for
the incidence of atherogenic vascular diseases often reported in
b-thalassaemic patients (Butthep et al, 1995). On the other
hand, very little is known about the effect of the a globin gene
mutation on the redox balance of individuals with a-thalas-
saemia. So far, no systematic study on redox status of
a-thalassaemic patients has been conducted.
In the present study, we demonstrate that a-thalassaemic
individuals were deficient in plasma antioxidants, and suffered
from higher levels of oxidative stress. The deficit of overall
antioxidant capacity correlated with the severity of the form of
a-thalassaemia. The a-thalassaemic erythrocytes, with crippled
antioxidant defence, showed increased lipid peroxidation of
their cell membrane. Moreover, these cells were more
susceptible to oxidant-induced lipid peroxidation and decrease
in deformability. Taken together, our experimental findings
suggest that the redox status is altered in a-thalassaemic
subjects, and this change may be implicated in the patho-
physiology of a-thalassaemia, particularly, in the elderly
thalassaemic individuals.
Materials and
methods
Chemicals
Unless otherwise stated, all chemicals were obtained from
Sigma Chemical Company (St Louis, MO, USA).
Patients
A total of 182 a-thalassaemic individuals and 50 healthy
volunteers were enrolled in the present study. Eighty-seven
Determination of vitamin A, E, and C in plasma
A method modified from that of Russel et al (1986) was used
for determination of plasma vitamin A and vitamin E (Cheng
et al, 1995). Briefly, 0Æ2 ml of d-tocopherol (1 lg/ml in
absolute ethanol) was added to an equal volume of plasma
while vortexing. After vortexing for 10 s, 1 ml of hexane was
added. The mixture was centrifuged at 1000 g for 5 min, and
the hexane layer was transferred to a vial for high performance
liquid chromatography (HPLC) analysis using the Waters
Alliance 2690 system (Waters, Milford, CA, USA). A lBondpark
column was used. The mobile phase was methanol and
H2O (95:5, v/v) and the flow rate was 1Æ6 ml/min. Vitamin A
was detected by an ultraviolet detector at 325 nm. Vitamin E
was detected using a fluorescence detector with excitation and
emission at 290 and 330 nm, respectively.

4. Determination of vitamin A, E, and C in plasma
A method modified from that of Russel et al (1986) was used
for determination of plasma vitamin A and vitamin E (Cheng
et al, 1995). Briefly, 0Æ2 ml of d-tocopherol (1 lg/ml in
absolute ethanol) was added to an equal volume of plasma
while vortexing. After vortexing for 10 s, 1 ml of hexane was
added. The mixture was centrifuged at 1000 g for 5 min, and
the hexane layer was transferred to a vial for high performance
liquid chromatography (HPLC) analysis using the Waters
Alliance 2690 system (Waters, Milford, CA, USA). A lBondpark
column was used. The mobile phase was methanol and
H2O (95:5, v/v) and the flow rate was 1Æ6 ml/min. Vitamin A
was detected by an ultraviolet detector at 325 nm. Vitamin E
was detected using a fluorescence detector with excitation and
emission at 290 and 330 nm, respectively.