EFFECT OF DIFFERENT PH BUFFERS AND DIFFERENT
TEMPERATURES ON LITHIUM CARBONATE-GLUTATHIONE
INTERACTION
Hashmat Ullah1, Muhammad Farid Khan1 & Farwa Hashmat 2
1
Department of pharmaceutical Chemistry, Faculty of Pharmacy Gomal University,
Dera Ismail Khan
1
Department of Chemistry, Gomal University, Dera Ismail Khan
ABSTRACT
Physiological pH and body temperature are both very important in the life of an organism because
different bio-chemical reactions take place at specific pH as well as on specific temperature. The rate
of a particular bio-chemical reaction may increase or decrease due to slight alteration in either pH or
temperature or both. Keeping in view this fact, we have studied the affect of lithium carbonate on
GSH chemical status (reduced glutathione) at six different pH and at three different temperatures by
using Ellman’s modified method. Our results show that lithium carbonate shows better interaction at
pH 7.5 and temperature 35oC which are both near to physiological pH and body temperature i-e
lithium carbonate cause decrease in GSH concentrations near the physiological pH and near body
temperature which may be due to the formation of Li-S-G complex or GSSG.
Keywords: Physiological pH, Temperature, Lithium Carbonate (Li2CO3), GSH (Reduced
Glutathione)
INTRODUCTION
many reactive metabolites by spontaneous
During the process of evolution, O2
conjugation or by a reaction which is
tension increases in the environment as a
catalyzed by the GSH S-transferases is the
result aerobic organisms need a strong
main role of reduced form of glutathione.
system for restoring sulfhydryl group to
(Coles et al., 1990; Hinchman
reduced form after exposure to oxidation
1994). Reduced form of glutathione has
stress.(Fridovish, 1989; Naqui
et al.,
strong affinity for metals and it acts as
1986), without a process to reduce protein
metal binding ligands as this form of
disulfides, cysteinyl residues of essential
glutathione has very important role in
enzymes might remain oxidized, leading
transport, storage and metabolism of
to changes in catalytic activity, this
metals. Glutathione is found in millimolar
function is full filled by the thiol-disulfide
concentrations in
exchange catalyzed by thiol-transferases in
2000), brain is very sensitive to oxidative
the presence of GSH (Sohal et al., 1996;
damage than rest of the body tissues
Sundquist et al., 1989). Detoxification of
(Halliwell
et al.,
Gomal University Journal of Research, 28(1). June, 2012
et al.,
brain (Dringen et al.,
1986) while GSH
2
Hashmat et al., Effect of Different pH
depletion weakens the defense system of
behavioral
responses
body and hence cause progression of
chlinergically mediated. Thus, lithium
disease. Administration of reduced GSH
reduces the convulsant threshold to the
showed improvement in patient with
nonspecific cholinergic muscarinic agonist
PD.(Sechi et al., 1996).
are coline and to anticholinesterases (Jope,
Serotonergic neurotransmission increases
2001).
when lithium is used for short term (Price,
decreases sensitivity of CNR (Central
et al., 1990; Shiah, et al., 2000) but this
nicotinic receptors) (Want et al., 1997).
Lithium
that
are
administration
also
effect is not observed after long-term
treatment and therefore the implications of
MATERIALS AND METHODS
this in the mechanism of action of lithium
in long term treatment and in its anti-
MATERIALS
suicide effect are unclear (Lenox, et al.,
Reduced
1995).Dopaminergic system can also be
Lithium
effected
Sodium
by
lithium.
neurotransmission
is
Dopamine
shown
to
be
glutathione
carbonate
(GSH)
(BDH,
hydroxide,
(Fluka),
Germany),
DTNB
Dithiobis-2-Nitrobenzoic acid
i-e5,5(Sigma),
decreased during acute administration of
KH2PO4 (Merck), Double refined distilled
lithium which may be link to lithium’s anti
water (D/W), HCl 35% (Kolchlight),
manic effect. Consistent turnover of
Oven: Memmert Model U-30 854 (Schwa
chronic
lithium
is
Boch, Germany), UV-Spectrophotometer
Behavioral
&
(UV-1601 Shimadzu, Japan), Magnatic
HIDRS
stirrer (Hungary), Micropippete 200 µl,
(Haloperidol induced dopamine receptor
500 µl, 1000 µl (Finnpipette Digital
supersensitivity) can not be prevented by
Finland),
chronic lithium treatment. (Lenox et al.,
Electrical balance type AX200 (Shimadzu
2000).Chronic
administration
Corporation, Japan), pH Meter: model
produces an up regulation of muscarinic
Nov-210 (Nova scientific Company Ltd,
receptors in the rat brain (De Bruin et al.,
Korea). All chemicals used in this research
2000). Studies have also shown that
were high quality chemicals and were used
lithium treatment enhances various
as purchased without further purification.
however
administration
very
biochemical
little.
symptoms
lithium
of
of
Disposable
Gomal University Journal of Research, 28(1). June, 2012
rubber
gloves,
3
Hashmat et al., Effect of Different pH
METHODS
26.66 µM, 40.00 µM, 53.33 µM, and
Preparation of Stock Solutions:
66.66 µM respectively starting from lower
15.4mg of GSH (reduced glutathione) was
used concentrations of GSH to higher used
added in 50ml of 0.2M buffer (phosphate)
concentration of GSH. From each GSH
pH7.6 to prepare 1mM GSH solution
dilution 0.2ml was mixed with 2.3 ml of
while a few drops of 0.1 N HCl were also
buffer (phosphate) having pH 7.6 then
added to it and the prepared solution was
500µl DNTB was mixed to this and these
kept in refrigerator till further use. 1ml of
mixtures were incubated for five minutes.
1mM GSH stock solution was taken and
After five min:, ABS( absorbance )of each
diluted with 9ml phosphate buffer pH 7.6
sample was recorded at fixed wavelength
to prepare 10ml of 0.1mM GSH solution.
λ max: 412 nm.
1mM DTNB solution was prepared by
DTNB Blank
dissolving 19.8mg of DTNB in 50ml
Solution of DTNB blank was prepared by
phosphate buffer pH 7.6. 1mM Li2O3
mixing 500µl of DTNB solution (1mM
solution was prepared by dissolving
DTNB) with 2500 µl of buffer (phosphate)
.369mg in 50ml distil water. To prepare
pH 7.6 and ABS ( absorbance) of DTNB
0.1mM lithium carbonate (Li2O3) solution,
blank solution was also recorded at λ max:
1.0ml of 1mM Li2CO3 stock solution was
412 nm.
taken into a test tube and was diluted with
Real Absorbance
distil water up to 10ml.
By subtracting absorbance of DTNB blank
Standard Curve
from the absorbance of each sample
Standard curve was drawn by preparing
(containing
four dilutions from 1mM GSH (Reduced
absorbance of each sample was obtained.
form of Glutathione) while 5th dilution was
All the obtained ABS were converted into
1.0mM GSH solution itself. The dilutions
GSH concentration by using equation
were in sequence of 200µM, 400µM,
given below and a standard curve was
600µM,
1000µM.
obtained
reduced
Equation
800µM
and
Concentration
(Final)
of
glutathione in
dilutions was 13.33µM,
GSH
+
y = mx + b
Gomal University Journal of Research, 28(1). June, 2012
DTNB),
real
4
Hashmat et al., Effect of Different pH
Fig 1: Standard Curve for Glutathione (GSH)
Affect of pH6.5, pH7.5, pH8.5, pH9.0,
recorded against reference cell containing
pH9.5, pH10.0 Buffer Solutions on
2.8ml buffer and 0.2ml of 0.1mM GSH at
Lithium
fixed wavelength λ max 412nm. As each
Carbonate-Glutathione
Interaction
time sample mixture was in different pH
pH6.5, pH7.5, pH8.5, pH 9.0, pH 9.5,
buffer so reference cell also contain buffer
pH10.0 buffer solutions were prepared and
of same pH as in sample cell. Control
2.3ml from each buffer was taken and
solution
separately mixed with 2ml of 0.1mM
glutathione was also prepared by taking
glutathione solution in six test tubes, then
4.3ml of each buffer solution in six test
1.0ml
5,5-dithiobis-2-
tubes separately. 2.0ml of 0.1mM solution
nitrobenzoic acid i-e DTNB was added to
of glutathione was added to test tube
this, all these six mixture were well shaken
following by the addition of 1.0ml of
for 10 minutes and to each of the six
1.0mM DTNB. After 5 minutes 3.0ml
mixtures
lithium
from each test tube was taken in cuvettes.
carbonate (Li2CO3) was added. These
GSH final concentration in control sample
mixtures were again very well shaken and
was 27.4µM (0.027mM) as in the sample.
incubated for 5 minutes. After 5 minutes,
ABS( Absorbances) were recorded after
3ml from each mixture was taken into
five minutes at fixed wavelength λ max
sample curette turn by turn and ABS was
412nm against reference cell containing
of
1mM
2.0ml
of
0.1mM
(glutathione
Gomal University Journal of Research, 28(1). June, 2012
blank)
for
5
Hashmat et al., Effect of Different pH
2.8 ml of respective phosphate buffers and
ABS( absorbances) which were then
0.2 ml of 0.1mM glutathione. Effect of
converted into conce: of glutathione
Li2CO3 on the chemical status of GSH
(Table 1) in mixture by well known
(reduced
glutathione) in pH6.5, pH7.5,
modified Ellman’s method as described
pH8.5, pH9.0, pH9.5, pH10.0 buffer
in standard curve for GSH( reduced
solutions was studied by determining the
glutathione).
Table #1:
Effect of pH6.5, pH7.5,pH8.5,pH9.0,pH9.5,pH10.0) Buffer Solutions on chemical Status of Glutathione (GSH) with
and without the presence of Lithium Carbonate
ABS i-e absorbance of 5,5-Dithiobis-2-Nitrobenzoic acid (DTNB) Blank solution was 0.057 ABS at fixed wavelength 412nm
Final Mixture contained GSH concentration 27.3µM
Real abs*/conc. of
GSH after reaction
ABS(Real)*/conc.
Mean of 3
with Lithium
of GSH Blank
Readings
Carbonate
Lithium
S#
pH
Carbonate
ABS
ABS
final conce:
st
nd
(1 )
(2 )
ABS
rd
(3 )
in Mixture
Abs.
Conc. of
GSH(µM)
Abs.
Conc. of
GSH(µM)
1
6.5
27.3 µM
0.481
0.478
0.475
0.478
0.421
2.72
0.761
4.89
2
7.5
27.3 µM
0.444
0.443
0.440
0.442
0.385
2.49
0.745
4.78
3
8.5
27.3 µM
0.454
0.452
0.447
0.451
0.394
2.55
0.753
4.83
4
9.0
27.3 µM
0.494
0.488
0.485
0.489
0.432
2.79
0.767
4.92
5
9.5
27.3 µM
0.512
0.509
0.507
0.509
0.452
2.92
0.772
4.96
6
10
27.3 µM
0.532
0.529
0.526
0.529
0.472
3.04
0.785
5.04
* ABS(Real)= ABS of Mixture - ABS of DTNB blank Solution.
Gomal University Journal of Research, 28(1). June, 2012
6
Hashmat et al., Effect of Different pH
Figure 2: Effect of different pH buffers on Lithium Carbonate-GSH interaction in aqueous medium
Effect of different pH buffers on lithium carbonate GSH interaction.
GSH control. Results are the mean ±SE of 3 experiments.
Affect of Different Temperatures i-e 25
0.5 ml of 1mM DTNB stock solution was
Degree Celsius, 35 Degree Celsius, 45
added into each test tubes (sample
Degree Celsius) on Lithium Carbonate-
mixture)
GSH Interaction
glutathione and lithium carbonate in the
2.0ml
of
concentration
of
sample test tube was 0.0033mM (3.33µM)
solution was added separately to 2ml of
respectively. Control solution (glutathione
0.1mM glutathione taken in three separate
blank) for glutathione was also prepared
test
(reaction
by taking 2ml of 0.1mM glutathione stock
mixtures) were well shaken and were kept
solution in a test tube to which 2ml of
in water bath for 10 minutes to maintain
phosphate buffer having pH 7.6 was
the temperature of (25 degree Celsius,
added.
35oC, 45 degree Celsius). Glutathione and
glutathione in control solution was also
lithium carbonate final concentration in
0.05mM (50µM) as in reaction mixture.
each of the reaction mixture was 0.5mM
0.2ml was taken from this sample and
(50µM) respectively.
2300µl (2.3 ml) of phosphate buffer
Sample cuvettes were prepared by taking
solution was mixed/added to it following
0.2ml of Li2CO3 plus glutathione mixture
by addition of 500µl of 1mM DTNB. The
from each one of the previously made test
ultimate final concentration of glutathione
tubes (reaction mixture) and diluted with
in
2.3ml of phosphate buffer pH 7.6 and then
(3.33µM). ABS (absorbance) was taken
These
lithium
final
carbonate
tubes.
0.1mM
the
mixtures
The
control
final
sample
Gomal University Journal of Research, 28(1). June, 2012
concentration
was
of
0.0033mM
7
Hashmat et al., Effect of Different pH
after five minutes at λ max 412nm against
temperature was studied in terms of
reference cell containing 2.8ml
buffer
determination of the absorbances which
solution (phosphate) having pH 7.6 and
were then converted into concentration of
0.2ml of 0.1ml glutathione solution. The
glutathione (Table2) in mixtures by a well
affect of lithium carbonate(Li2CO3)on the
known Ellman’s method, as mentioned in
chemical status of glutathione at different
standard curve for glutathione.
Table #2:
Effect of temperatures i-e 25 degree Celsius,35 degree Celsius,45 degree Celsius on chemical Status of Glutathione
(GSH) 0.1mM
ABS of 5,5-dithiobis-2-nitrobenzoic acid (DTNB) blank solution was 0.060ABS at fixed wavelength 412nm
In final Mixture GSH concentration is 3.33µM
Real abs*/conc. of
Final Conc.
S#
GSH after reaction
ABS(Real) */conc.
of GSH Blank
Temper
Of Lithium
ABS
ABS
ABS
Mean of 3
with Lithium
ature
Carbonate
1st
2nd
3rd
Readings
Carbonate
in Mixture
Abs.
Conc. of
GSH(µM)
Abs.
Conc. of
GSH(µM)
1
25 oC
3.33 µM
0.299
0.297
0.295
0.297
0.237
1.55
0.441
2.85
2
35 oC
3.33 µM
0.278
0.276
0.275
0.276
0.216
1.41
0.432
2.79
3
45 oC
3.33 µM
0.311
0.306
0.301
0.306
0.246
1.61
0.427
2.69
*ABS (Real) = ABS of Mixture - ABS of DTNB blank Solution.
Gomal University Journal of Research, 28(1). June, 2012
8
Hashmat et al., Effect of Different pH
Figure 3: Effect of different Temprature (25 degree Celsius, 35 degree Celsius, 45 degree Celsius) on lithium carbonate-glutathione
interaction in aqueous medium
Effect of different temperature on lithium carbonate GSH interaction.
GSH control. Results are the mean ±SE of 3 experiments.
RESULTS
the remaining concentration of GSH was
Affect of pH6.5, pH7.5, pH8.5, pH9.0,
left minimum (Table 1 and Fig1) in buffer
pH9.5, pH10.0 Buffer Solutions on
pH 7.5 indicating that this buffer with pH
Li2CO3-GSH Interaction in Aqueous
7.5 is favorable for interaction between
Medium.
Li2CO3 and GSH (near to body pH). Our
To study the affect of lithium carbonate
results show that although Li2CO3 reacts
(Li2CO3) on the chemical status of GSH
with GSH in different pH buffer but it
(reduced form of glutathione) in different
cause
pH buffers, we have prepared buffers of
concentrations in buffer pH 7.5 indicating
six different pH i.e. pH 6.5, pH 7.5, pH7.8,
that lithium in the same way might be the
pH 9.0, pH 9.5 and pH 10.0 and it was
cause of GSH depletion in human body.
found that Li2CO3 has best interaction in
The decrease of GSH in buffer pH 7.5
buffer pH 7.5 which is very close to
after the reaction with lithium carbonate
physiological pH of human body. After the
can
reaction of lithium carbonate ( Li2CO3 )
concentration in the same buffer of pH 7.5.
greater
be
depletion
compared
Gomal University Journal of Research, 28(1). June, 2012
from
in
its
GSH
control
9
Hashmat et al., Effect of Different pH
In buffer pH 7.5 the concentration of GSH
carbonate and GSH inside the human at
in control is 4.78 µM which has reduced to
normal body temperature 37oC. The
2.49 µM after the reaction of lithium
remaining GSH concentration at 35oC is
carbonate with GSH, shows a sufficient
1.41µM as compare to GSH control
reactivity of lithium with GSH at this pH
concentration
7.5.
temperature-total difference is 1.38µM
2.79
mM
at
this
when lithium carbonate and GSH both
Affect of Different temperatures i-e 25
were used in the ratio of 1:1 i-e 0.1mM of
degree Celsius, 35 degree Celsius, 45
lithium carbonate (Li2CO3) and 0.1mM of
degree
GSH (used concentration)
Celsius
on
Li2CO3-GSH
and final
Interaction in Aqueous Medium
concentration in sample mixture left
Different human body parts have slightly
3.33µM of each.
difference in there temperature but overall
temperature can fluctuate about one degree
(Fo) throughout the day. Accepted normal
human body temperature range is from
36.1oC to 37.2oC. Because of this fact, the
effect of
Li2CO3 on GSH ( Reduced
glutathione) at different temperature (25
degree Celsius, 35 degree Celsius, 45
degree Celsius) in aqueous medium was
studied. Our results as shown in table 2 &
Fig 3, shows that at 35oC there is greater
decrease in the concentration of GSH as
compared
to
carbonate
with
interaction
GSH
at
of
lithium
other
two
temperature i-e 25oC and 45oC. 35oC is a
temperature near to normal human body
temperature and greater decrease in GSH
concentration at this temperature indicates
greater chance of interaction of lithium
DISCUSSIONS
Human
body
physiological
temperature
pH
of
and
different
compartments of body are very crucial for
interaction of almost all medicines taken
by human beings for the treatment of
different
abnormalities
ailments/disease
including
the
and
under
consideration compound of lithium i-e.
Li2CO3 which is used for the treatment of
mania dipolar episodes and even for the
treatment of granulocytopenia and as an
adjuvant for cancer chemotherapy (Catan
et al, 1977). Lithium carbonate is being
used since long for the treatment of above
mentioned abnormalities/ diseases and in
addition to this still lithium is used for
many other disorder, it has also antisucidle
Gomal University Journal of Research, 28(1). June, 2012
10
Hashmat et al., Effect of Different pH
properties so it was a matter of interest to
study the affect of lithium carbonate at
metabolism and amphetamine induced
locomotor stimulation in rats. Journal of
Neural Transmission, P,239-250.
different pH buffers as well as at different
temperatures and our results show that
lithium carbonate-GSH interaction is more
at both (pH 7.5) near to body pH and
Bymaster F P & Felder C C (2002). Role
of cholinergic muscarinic system in
bipolar disorder and related mechanism of
action of antipsychotic agents. Molecular
Psychiatry, 7 (suppl. 1), S57-S63.
temperature near to body temperature. As
shown in table 1 Fig 2 and table 2 ,Fig 3.
CONCLUSIONS
Results show that lithium carbonateglutathione interaction is greater at pH 7.5
and temperature 35oC. Our study indicats
that lithium is also a cause of considerable
depletion in GSH concentration. Although
there are so many reasons to used lithium
carbonate for the treatment of many
human diseases/ abnormalities but this is
again a fact that lithium interact with
reduced glutathione (GSH) and depletes it.
Coles B & Ketterer B(1990). The role of
glutathione and glutathione transferases in
chemical carcinogenesis: Crit Rev
Biochem Mol Biol 25: Pp. 47-70.
De Bruin V M S, Marinho M M , & De
Sousa, F C F (2000). Behavioural and
neurochemical alternations after lithiumpilocarpine administration in young and
adult rats. A comparative study.
Pharmacology
Biochemistry
and
Behaviour, 65, 574-551.
Dringen RGJ & Hirlinger J (2000).
Glutatione metabolism in the brain: Eur J
Biochem. 267: Pp. 4912-4916.
Ellaman GL (1959). Determination of
sulfhydryl group Arch. Biochem. Biophys.,
89: 70-74.
Depletion of GSH may be due to the
formation of
Li-S-G complex formation or GSSG
formation.
Li2CO3 +2GSH
-1
GS +GS
-1
2Li-SG+H2CO3
GSSG
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