Vol.2, No.10, 1221-1225 (2010) Health
doi:10.4236/health.2010.210181
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Effect of calcium and diltiazem on
phenylhydrazine-induced oxidative injury in goat
erythrocytes
Kaushik Das*, Jharna Bhattacharyya
Division of Cell Biology and Physiology, Indian Institute of Chemical Biology, Kolkata, India; *Corresponding Author:
Received 8 June 2010; revised 22 June 2010; accepted 7 July 2010.
ABSTRACT
Lipid peroxidation, hemolysis and thiol contents
were studied in intact goat erythrocytes exposed
to phenylhydrazine. An increase in lipid peroxi-
dation, hemolysis and thiol content was observ-
ed after phenylhydrazine treatment of erythrocy-
te. Extracellular Ca2+ potentiates the phenylhy-
drazine-induced lipid peroxidation and hemoly-
sis of erythrocytes significantly. Ca2+ does not
influence much the thiol content of phenylhydr-
azine treated erythrocytes. No effect of Ca2+ on
control lipid peroxidation, hemolysis and thiol
contents of erythrocytes was observed. Diltiaz-
em and EDTA inhibited the increased responses
of lipid peroxidation and hemolysis due to Ca2+.
However the thiol content was not much influen-
ced by either diltiazem or EDTA. The results su-
ggest that oxidative damage of erythrocyte cau-
sed by phenyl hydrazine could be prevented by
calcium channel antagonist, diltiazem, which
may act as antioxidant also.
Keywords: Ca2+; Diltiazem; Erythrocyte; Free
Radical; Lipid Peroxidation; Phenylhydrazine
1. INTRODUCTION
Phenylhydrazine (PHZ) and other oxygen generating sy-
stems cause elevation of lipid peroxidation level in ery-
throcytes [1]. PHZ through oxidative stress also altered
methemoglobin level, catalase activity and turbidity of
erythrocyte [1], which are typical biochemical markers
of haemolytic anaemia. Oxidative stress is defined as str-
uctural and/or functional injury produced in tissues by the
uncontrolled formation of pro-oxidant free radicals. Oxi-
dative stress usually develops when pro-oxidant action
of an inducer exceeds the anti-oxidant capacity of the
cell-defence system, altering its homeostatic capacity.
PHZ, in presence of haemoglobin autooxidizes to form
hydrogen peroxide, which leads to hemolysis, resulting
in severe haemolytic anaemia [2] and generates reactive
oxygen species [3] among which hydroxyl radical, OH· [4]
is highly reactive and initiates the peroxidation of unsa-
turated fatty acids in endogenous phospholipids. Oxida-
tive stress due to overproduction of reactive species pl-
ays a significant role in the pathogenesis of various dis-
eases and involves numerous mechanisms. All biological
molecules are prone to free radical attack. Lipid peroxi-
dation is a chain reaction, involving numerous biprod-
ucts caused by free radical damage.
The modulators involved in the production of haemo-
lytic anaemia caused by PHZ are not fully understood.
Ca2+ has been implicated as an important contributory
factor to cell damage caused by the resultant effect of
oxidative stress in several diseases [5]. Ca2+ influx has
been reported to increase in pathophysiological condi-
tions where Ca2+-channel blockers have got an important
regulatory role [5]. In some studies the role of lipid per-
oxidation in cell death has implicated a concurrent invo-
lvement of Ca2+. The component, which increases Ca2+
influx-increases lipid production biproducts also. A nu-
mber of experimental systems have been used to exam-
ine the interaction between lipid peroxidation and Ca2+
as mediators of functional membrane damage [6]. Volt-
age dependant calcium channels (VDCCs) play a critical
role in the regulation of levels of intracellular Ca2+ and
thereby control an array of physiological processes in
cells and VDCCs functioning could be prevented by
using calcium channel blockers [7]. Thus regulating Ca2+
influx through VDCCs by using calcium channel block-
ers, which act as antioxidants as well, could have some
impact on the calcium mediated lipid peroxidation in
several disease conditions.
The present study is an attempt to study the role of ca-
lcium and calcium channel blocker diltiazem, on lipid

K. Das et al. / Health 2 (2010) 1221-1225
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
1222
peroxidation induced by PHZ in erythrocytes. Since in
haemolytic diseases membrane fragility is altered, the
hemolysis as well as the thiol content of erythrocytes was
also studied under the same experimental condition.
2. MATERIALS AND METHODS
All the experiments were performed using goat blood,
which was collected from acid-citrate dextrose solution.
The packed erythrocytes were isolated by centrifugation
at 3000 g for 10 min at 4ºC. The plasma and buffy coat
were removed by aspiration and the erythrocytes thus
obtained were washed thrice and suspended in 0.9%
NaCl solution [1].
Incubation of erythrocytes with calcium and calcium
channel blocker - The incubation medium contained 10
mM sodium phosphate buffer (pH 7.4), 1 mM PHZ, 2
mM Ca2+, 20 M diltiazem/2 mM EDTA, 10 mM Glu-
cose and erythrocyte suspension was incubated at 37ºC
in a shaker water bath. At the end of the incubation the
cells were collected by centrifugation, washed and lysed
with 5 mM sodium phosphate buffer (pH 8.0, 1:10 w/v)
and hemolysate were obtained by centrifugation at
10,000x g for 1 hr. The resulting supernatant was taken
for experimental purposes.
Lipid peroxidation: Lipid peroxidation of hemolys-
ates from both control and treated erythrocytes were de-
termined by malondialdehyde (MDA) estimation as done
previously [1]. Thio barbituric acid (TBA) reactant pro-
ducts were estimated by MDA liberated by the breakdo-
wn of polyunsaturated fatty acids and expressed as MD-
A/mg protein by calculating the extinction co-efficient
of MDA [8].
Hemolysis: After incubation of erythrocytes with the
other ingredients, equal volume of saline was added to the
incubation mixture and centrifuged at 2000 rpm for 10 min
and the supernatants were taken for absorbancy at 540 nm
against reagent blank [9]. During incubation same amo-
unt of packed cell proteins were added to each tube.
Thiol estimation: Thiol content of the hemolysates
was estimated according to Owens and Belcher [10]. Tr-
ichloroacetic acid (TCA) was used instead of metaphos-
phoric acid for protein precipitation. 0.5 ml of 5% TCA
filtered extract of each sample was added to a mixture of
1.5 ml of 0.5 M potassium phosphate buffer, pH 8.0 and
0.03 ml of dithionitrobenzene reagent. The optical dens-
ity was measured at 412 nm after 3 min and compared
with the standards made with reduced glutathione dissol-
ved in 0.5% TCA. Values are expressed as nmoles of th-
iols liberated per mg protein.
Protein measurement: The protein content of the sa-
mples was determined according to the method of Brad-
ford [11].
Statistical analysis: Statistical significance was mea-
sured as mean ± SD and Student’s t-test was used for
determination of statistical significance.
3. RESULTS AND DISCUSSION
Goat erythrocytes when incubated with PHZ (1 mM), a
strong oxidant, caused an elevation (P < 0.001) of lipid
peroxidation (Figure 1). The increase was about 126%
over control values. Additionally, in presence of divalent
cation Ca2+ (2 mM) there was further elevation of lipid
peroxidation (Figure 1). Below the concentration of 2 mM
Ca2+ with PHZ, any significant increase of lipid peroxi-
dation could not be detected, hence in our further exp-
eriments 2 mM concentration of Ca2+ was chosen as opti-
mum. Diltiazem, a calcium channel antagonist, at 20 µM
antagonizes the action of calcium (Figure 1) and hence
the elevated level of lipid peroxidation was suppressed
by the addition of diltiazem (P < 0.01, compared to PHZ
(1 mM) + Ca2+ (2 mM) treated group). Similarly, additi-
on of 2 mM EDTA (equimolar concentration of Ca2+) to
the incubation medium also caused inhibition of increased
lipid peroxidation caused by PHZ (1 mM) + Ca2+ (2 mM).
Like lipid peroxidation, the other criteria of PHZ ac-
tion were to check the change of hemolysis pattern of
erythrocyte. As shown in Figure 2, it is evident that the
hemolysis of erythrocyte occurred significantly when in-
cubated with PHZ (0.16 vs. 0.02). Ca2+ elevated the level
further, diltiazem and EDTA suppressed the haemolysis
values due to PHZ and Ca2+ (Figure 2).
The protein degradation product, the thiol content, was
found to alter when erythrocytes were treated like before
(Table 1). As shown in Table 1, when erythrocytes were
Figure 1. Effect of Ca2+ diltiazem and EDTA on
PHZ-induced lipid peroxidation of erythrocytes. [A:
Control erythrocytes without any treatment; B: Control
erythrocytes with 2 mM Ca2+; C: 1mM PHZ-treated
erythrocyte, P < 0.001 against A; D: 1 mM PHZ + 2 mM
Ca2+, P < 0.001 against C; E: 1 mM PHZ + 2 mM Ca2+ +
20 µm diltiazem, P < 0.01 against D; F: 1 mM PHZ + 2
mM Ca2+ + 2 mM EDTA, P < 0.05 against D. Values
given are mean ± SD of 6 different experiments]. * P <
0.001, ** P < 0.01, *** P < 0.01, # P < 0.05.

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Figure 2. Effect of Ca2+, diltiazem and EDTA on PHZ-induced
hemolysis of erythrocyte. [A: Control erythrocyte without any
treatment; B: Control erythrocytes with 2 mM Ca2+; C: 1mM
PHZ-treated erythrocyte, P < 0.001 against A; D: 1 mM PHZ +
2 mM Ca2+, P < 0.001 against C; E: 1 mM PHZ + 2 mM Ca2+ +
20 µM diltiazem, P < 0.01 against D; F: 1 mM PHZ + 2 mM
Ca2+ + 2 mM EDTA, P < 0.05 against D. Values given are
mean ± SD of 6 different experiments]. * P < 0.001, ** P <
0.01, *** P < 0.01, # P < 0.05.
Table 1. Effect of Ca2+ and diltiazem and EDTA on thiol con-
tent of PHZ treated erythrocyte.
[Values are means ± SD of 6 different experiments]
System Thiol content
(n mol/mg protein)
Control 28.79 ± 0.23
Control + Ca2+ (2mM) 27.55 ± 0.22
PHZ (1mM) 32.00 ± 0.18*
PHZ (1mM) + Ca2+ (2mM) 37.58 ± 0.22**
PHZ (1mM) + Ca2+ (2mM) + diltiazem (20µm) 30.48 ± 0.15***
PHZ (1mM) + Ca2+(2mM) + EDTA (2mM) 30.50 ± 0.14#
*P < 0.001, **P < 0.01, ***P < 0.01, #P < 0.05
treated with PHZ (1 mM) alone, the thiol content was
increased and addition of Ca2+ (2 mM) to the system fur-
ther enhanced the thiol level. In comparison to lipid pe-
roxidation and hemolysis, the increment observed was
less when thiol was measured from treated erythrocytes.
Both diltiazem and EDTA were found to reduce the in-
creased level of thiols in PHZ and Ca2+ treated erythro-
cytes (Table 1). Further we have confirmed our lipid pe-
roxidation experiment using Cu2+ (0.2 mM) + Ascorbic
acid (1 mM) system (Table 2). Results obtained showed
that the changes observed were more or less similar in
Cu2+-Ascorbate system as compared with PHZ.
The involvement of oxygen-derived free radicals and
other oxidant species in numerous pathophysiological co-
nditions helps to develop suitable systems to study their
effects and also to assess the antioxidant activity of en-
do/exogenous compounds. To confirm the previous re-
sults [1], the effect of modulators was evaluated on PHZ
treated erythrocytes. An erythrocyte model was taken for
lipid peroxidation, hemolysis and thiol content, where
PHZ was chosen as oxidant and Ca2+, diltiazem and ED-
TA as modulators of PHZ mediated action. PHZ gener-
ally produces haemolytic anaemia in in vivo system. The
present results indicate that the damaging effect due to
PHZ on erythrocyte could be repeated in in vitro system
also. Ca2+ could have an important role in the pathoph-
ysiology of sickling process. Sickle cells, which main-
tain an abnormally deformed shape, have three to seven
times higher Ca2+ level as compared to normal red blood
cells [12]. Interestingly the results have shown that Ca2+
enhanced the action of PHZ on lipid peroxidation and
hemolysis mostly. These two criteria are often observed
in anaemic condition. Lipid peroxidation, a marker of
membrane function, as well as hemolysis is greatly al-
tered when Ca2+ is added with PHZ to the incubation
medium. Ca2+ alone without PHZ, on the other hand has
got no significant effect on the criteria studied above.
Many of the mechanical properties of sickle cell me-
mbrane have been found to relate to high Ca2+ level [13].
Because of the role of Ca2+ in injurious processes, Ca2+
antagonists have been tested for reducing the detrimental
consequences. In vitro studies on Ca2+ antagonists have
been carried out by various workers [14] with the view
to prevent the injurious effects of Ca2+ on erythrocytes.
They concluded that these Ca2+ antagonists either singly
or in combination might be useful in preventing vaso-
occlusive events. Ca channel antagonists were shown to
act as antioxidant [15]. Calcium antagonists antioxidant
potency correlated directly with the compound’s relative
affinity for the membrane lipid bilayer [16]. Similarly in
Table 2. Effect of Ca2+ diltiazem and EDTA on Cu2+-ascorba-
tea-induced lipid peroxidation of erythrocytes.
[Values are means ± SD of 6 different experiments]
System Lipid peroxidation
(n mol /mg protein)
Control 168.25 ± 1.67
Control + Ca2+ (2 mM) 170.55 ± 1.01
Cu2+ (0.2 mM) + Ascorbic acid (1 mM) 335.00 ± 4.23*
Cu2+ (0.2 mM) + Ascorbic acid (1 mM) +
Ca2+ (2 mM) 398.65 ± 3.67**
Cu2+ (0.2 mM) + Ascorbic acid (1 mM) +
Ca2+ (2 mM) + diltiazem (20 µm) 305 ± 2.67***
Cu2+ (0.2 mM) + Ascorbic acid (1 mM) +
Ca2+ (2 mM)+ EDTA (2 mM) 346 ± 4.33#
*P < 0.001, **P < 0.01, ***P < 0.01, #P < 0 .05
aAssay system was followed according to Halder, J. and Bhaduri, A.N.,
1998 [3].

K. Das et al. / Health 2 (2010) 1221-1225
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
1224
our studies the results have shown that micromolar conc-
entration of Ca2+ antagonist, diltiazem, decreased the le-
vel of lipid peroxidation, hemolysis and thiol content of
erythrocyte when treated with PHZ and Ca2+. Metal che-
lator EDTA, on the other hand was not found to be as ef-
fective as diltiazem in preventing these damaging proc-
esses.
Earlier reports [17] using erythrocyte as a model have
shown increased erythrocytic protein degradation when
erythrocytes were treated with PHZ. Therefore, it is exp-
ected that more the degradation, more thiol groups wou-
ld be liberated. Practically this was found in the present
experiments, where in vitro addition of PHZ plus Ca2+
caused increased SH level (though not to a greater extent)
and diltiazem was as effective as before. EDTA could
not markedly prevent the degradative phenomenon cau-
sed by PHZ plus Ca2+.
Lipid peroxidation in biological membranes causes
impairment of membrane function [18] decreases fluid-
ity and inactivation of membrane bound enzymes and
receptors [19]. In most cases haemoglobin acts as the ca-
talyst for lipid peroxidation [4] and the erythrocyte me-
mbrane becomes an important target for damage [20]. Su-
peroxide and hydroxyl radical might participate in PHZ-
induced toxicity [2]. Administration of PHZ to animals
can induce peroxidative lipid damage of erythrocyte me-
mbrane resulting in the accumulation of MDA [21]. Ma-
ny toxicants like PHZ induce hemolysis by the alteration
of cytoskeletal protein [22]. Further detailed examina-
tion is necessary to clarify the mechanism of action of
PHZ. Results from the present experiments suggest that
selecting erythrocyte, as an in vitro model, could be used
to ascertain the mechanism of haemolytic anaemia and
prevention by calcium channel blocker which could act
as antioxidant.
4. ACKNOWLEDGEMENTS
Thanks are due to the Council of Scientific and Industrial Research
(CSIR), New Delhi, for the award of a research fellowship to Kaushik
Das. We also acknowledge Late Dr. Asoke G Datta for his valuable
advice and constructive criticism till the last moment of his life.
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