GSH (g-glutamylcysteinylglycine) is a sulfhydryl (-SH) antioxidant, antitoxin, and enzyme
cofactor. Glutathione is found in animals, plants, and microorganisms, and due to its water
solubility is mainly found in the cell cytosol and other aqueous phases of the living system
(Kosower and Kosower, 1978; Kidd, 1991; Lomaestro and Malone, 1995; Meister, 1976).
Glutathione exists in two forms: the antioxidant "reduced glutathione" tripeptide is
conventionally called glutathione and abbreviated GSH; the oxidized form is a sulfur-sulfur
linked compound, known as glutathione disulfide or GSSG. The GSSG/GSH ratio may be a
sensitive indicator of oxidative stress.
GSH has potent electron-donating capacity, as indicated by the high negative redox potential of
the GSH/GSSH "redox couple" (E'0 =-0.33v) (Lewin, 1976). Its high redox potential renders
GSH both a potent antioxidant and a convenient cofactor for enzymatic reactions that require
readily available electron pairs (Kehrer and lund, 1994). The reducing power of GSH is a
measure of its free radical scavenging, electron-donating, and sulfhydryl-donating capacity.
The reduced glutathione molecule consists of three amino acids - glutamic acid, cysteine, and
glycine - covalently joined end-to-end. The sulfhydryl (-SH) group, which gives the molecule its
electron-donating character, comes from the cysteine residue.Glutathione is present inside cells
mainly in its reduced (electron-rich, antioxidant) GSH form. In the healthy cell GSSG, the
oxidized (electron-poor) form, rarely exceeds 10 percent of total cell glutathione (Kosower and
Kosower, 1978). Intracellular GSH status appears to be a sensitive indicator of the cell's overall
health, and of its ability to resist toxic challenge. Experimental GSH depletion can trigger suicide
of the cell by a process known as apoptosis (Duke et al., 1996; Slater et al., 1995). Aluminium
compounds are used in many diverse and important industrial applications such as alums in
water-treatment and alumina in abrasives and furnace linings. They are found in consumer
products such as antacids, astringents, buffered aspirin, food additives, and antiperspirants.
Powdered Aluminium metal is often used in explosives and fireworks (Sidney, 2007). The use of
Aluminium cookware, popular because of its corrosion resistance and good heat conduction, has
not been shown to lead to Aluminium toxicity in general. Excessive consumption of antacids
containing Aluminium compounds and excessive use of Aluminium-containing antiperspirants
are more likely causes of toxicity. In research published in the Journal of Applied Toxicology,
Dr. Philippa D. Darby of the University of Reading has shown that Aluminium salts increase
estrogen-related gene expression in human breast cancer cells grown in the laboratory. These
salts estrogen-like effects have lead to their classification as a metalloestrogen. It has been
suggested that Aluminium is a cause of Alzheimer's disease, as some brain plaques have been
found to contain the metal. Aluminium metal has affinity for the glutathione present in aqueous
phases of blood. This affinity is mainly formed between Aluminium metal and sulfhydryl groups
of glutathione (Quig, 1998). This affinity can cause a depletion of the reduced form glutathione
in the blood, but with the depletion of the glutathione, GSH synthesizing systems start making
more GSH from cysteine via the -glutamyl cycle but if GSH is usually not effectively supplied,
however, if GSH depletion continues because of chronic metal exposure (Quig, 1998; Hultberg
et al., 2001 Stohs and Bagchi,1993) then the pharmacological benefits of the Aluminium metal
being used for the help of body defenses can be harmful in nature to the body defense system .
The following study makes a design to see the effects of Aluminium Sulphate, in respect of
concentration and time, on glutathione level in Plasma.
MATERIALS AND METHODS
Sodium Hydroxide (Fluka AG), L.Glutathione (GSH)(Fluka), DTNB (Sigma), Potassium
Dihydrogen Phosphate (Merck), HCl 35% (Kolchlight) Aluminium Sulphate (Merck,Germany),
Sodium Chloride (Merck), Disodium Edetate (Riedel Dehean AG Sleeze Hannover) Chloroform
(Merck), Ethanol(Merck). Distilled Water (Double Distilled). U.V 1601 spectrophotometer
(Shimadzu). Centrifuge (H-200). PH Meter: Model NOV-210, Nova Scientific Company Ltd.
Korea, Oven: Memmert Model U-30,854 Schwabach (Germany). Magnetic Stirrer, hot plate
400(England). Micropipettes 200 µl, 500 µl, 1000 µl were used of Socorex Swiss (Finaland),
Sortorius Balance, Disposable Rubber Gloves, were used in this research work.
METHODS (Ellman’s, 1959)
Isolation of Plasma
Sample of 5 ml of human venous blood treated with heparin to prevent clotting was collected.
The blood was centrifuge on H-200 centrifuge at 10,000 rpm for 2 minutes. The plasma was
removed with Pasteur pipette. One ml of plasma was incubated for different concentration and
time interval with I ml of metal, and analyzed for GSH level.
Determination of GSH in Plasma
The assay of GSH with DTNB was performed followed a standard Ellman’s method for plasma
of blood. 2.3 ml of potassium phosphate (0.2 M, pH 7.6) buffer was taken in the cell and/or
cuvete followed addition of 0.2ml aqueous solution or plasma of blood. To it 0.5 ml DTNB
(0.001M) in a buffer was added. An absorbance of reaction product in cuvette was read after 5
minutes at 412 nm using shamadzo 1601 UV/Visible double bean spectrophotometer and GSH
level was determined, from standard curve of reduced GSH obtained with 0.2, 0.4, 0.6, 0.8 and
1mM GSH concentration.
Standard Curve for Glutathione
200µl of 0.2, 0.4, 0.6, 0.8 and 1mM solutions of glutathione was added to 2.3ml of phosphate
buffer pH 7.6, followed by the addition of 0.5ml of 1mM DTNB Stock solution. The mixtures
were shaken thoroughly and incubated for 5 minutes at 300C. Absorbances were taken after 5
minutes at fixed wavelength of 412nm.
Blank was prepared in which GSH was omitted. Standard curve was constructed by plotting the
change of absorbance versus final concentration of GSH in the mixture. Straight line was drawn
by using linear regression analysis. The correlation coefficient of plot was 0.999. Standard curve
was obtained as shown in the figure 1.
Effect of Different Concentrations of Aluminium Sulphate on Glutathione (GSH) level in
Plasma of Human Blood
To 1ml (1000ml) of plasma taken in five separate test tubes, 1ml (1000ml) of different
concentrations of 0.2, 0.4, 0.6, 0.8 and 1mM solution of Aluminium Sulphate were added
separately and shacked. Five separate test tubes was prepared with 0.2ml (200ml) Aluminium
Sulphate plus plasma mixture from each previously made five tubes diluted with 2.3ml (2300ml)
of phosphate Buffer pH 7.6 and added 0.5ml (500ml) of 1mM DTNB stock solution. A control
for plasma was also prepared by taking 1ml (1000ml) of plasma in a test tube and diluted with
1ml (1000ml) of phosphate buffer pH 7.6.The effect of Aluminium sulphate on the chemical
status of glutathione in plasma was studied in terms of determination of concentration of GSH in
mixtures by a well known Ellman’s method, as mentioned in standard curve for GSH. The
concentrations of GSH were determined from the GSH standard curve.
Effect of Aluminium Sulphate on Glutathione (GSH) level in Plasma with Time
To 1ml (1000ml) of Plasma taken in a test tube, 1ml (1000ml) of 1mM solution of Aluminium
Sulphate was added and shacked. The final concentration of Aluminium sulphate was 0.5mM
(500mM). A test tube with 0.2ml (200ml) Aluminium sulphate plus plasma mixture was prepared
from previously made test tube diluted with 2.3ml (2300ml) of phosphate buffer pH 7.6 and
added 0.5ml (500ml) of 1mM DTNB stock solution. The final concentration of Aluminium
sulphate was 0.03333mM (33.33mM).A control for plasma was also prepared by taking 1ml
(1000ml) of plasma in a test tube and diluted with 1ml (1000ml) of phosphate buffer pH 7.6.The
effect of Aluminium sulphate glutathione level in plasma was studied in terms of determination
of concentration of GSH in mixtures by a well known Ellman’s method, as mentioned in
standard curve for GSH. The absorbances were read at 0, 30, 60, 90, 120, 150 minutes after
preparing mixture (1ml of plasma plus 1ml of Aluminium sulphate).The concentrations of GSH
in plasma were determined from the glutathione standard curve.
Effect of Aluminium Sulphate on Glutathione (GSH) level in Plasma
Effect of Aluminium metal on the chemical status of glutathione present in plasma was studied
in term of determination of concentration of glutathione. Aluminium metal caused a decrease in
the concentration of glutathione present in plasma. Different concentrations of Aluminium cause
a gradual decrease in the concentration of glutathione in plasma as the concentration of metal
increased as shown figure 2 and table 3.
Effect of Aluminium on the chemical status of glutathione was also studied for the time
dependency and noted that the concentration of glutathione was gradually decreased as the time
passes from 0minute interval of time to 150 minutes as shown figure 3 and table 4.
Statistical Analysis for Effect of Aluminium Sulphate Glutathione (GSH) Level in Plasma
Statistical approach for the effect of Aluminium sulphate on the chemical status of GSH was also
conducted for the concentration and time dependent effects. The paired comparison T-test (Table
5) of concentration dependent effect of Aluminium sulphate and GSH blank gave the decision
that there is an effect of Aluminium on GSH level in plasma with increase in concentration of
Aluminium sulphate, as compared to GSH blank solution treatment.
Similarly the paired comparison T-test (Table 6) of time dependent effect of Aluminium sulphate
and GSH blank gave the decision that there is an effect of Aluminium sulphate on the chemical
status of GSH in plasma as the passage of time is increased with a specific concentration of
Aluminium sulphate as compared to GSH blank solution treatment.
There is increasing interest in glutathione due to its varied physiological and pharmacological
properties including detoxification through participation in the redox system, activation of SHEnzymes,
co-enzymatic action and conjugation. Aluminium has been found to play a role in
apoptosis (gene-directed cell death), a critical cellular regulatory process with implications for
growth and development, as well as a number of chronic diseases. Cells in the salivary gland,
prostate, immune system and intestine can secrete Aluminium. Thus it was of interest to study
the interaction of this metal in vivo to establish further scientific data. This scientific data about
the interaction and the effect of Aluminium sulphate on the chemical modulation of GSH will
enable us to understand further the role of, Aluminium sulphate and GSH and strengthen our
knowledge about their therapeutic uses in many diseases. Different concentrations of Aluminium
caused decrease of concentration of glutathione and play important role in the conversion of
GSH to either GS Al or GS-Al of reduced form SG in plasma. The effect of Aluminium Sulphate
was studied for the concentration and time dependent effects on the glutathione level and was
found that the concentration of reduced glutathione was decreased with increasing concentration
of Aluminium metal in solution and with the passage of time respectively. The following
sequences of reactions are suggested to be happened in the experiment.
GSH + Aluminium (Al) Al (GS)3
The results also suggested that there was a possibility of formation of intermediate or conjugate
between Aluminium and GSH. However it was not possible to estimate or determined those
conjugates under those conditions .Since both GSH and Aluminium, is biological active
compounds. It was of interest to study the possible interaction of this metal in vitro as a model of
in vivo interaction.
The tripeptide thiole glutathione has facile electron-donating capacity, linked to it sulfhydryl
(_SH) group. Glutathione is important water phase antioxidant and essential cofactor for
antioxidant enzyme. It provides protection also for the mitochondria against endogenous
radicals. Its high electron donating capacity combined with its high molecular concentration
endows (GSH) with great reducing power, which is used to regulate a complex thiole- exchange
Different concentration of Aluminium metal caused a gradual decreased in the concentration of
glutathione in plasma. Effect of Aluminium on the chemical status of glutathione was also
studied for the time dependency and noted that the concentration of glutathione gradually
decreased as the time passes from 0 minute interval of time to 150minutes in plasma.
Duke RC, Ojcius DM and Young JDE (1996). Cell suicide in health and disease. Scientific
Ellman GL (1959). Determination of sulfhydryl group.Arch.Biochem.Biophys.; 82: 70-74
Hultberg B, Andersson A and Isaksson (2001). Interaction of metals and thiols in cell damage
and Glutathione distribution: potentiation of mercury toxicity by dithiothreitol. Tox. 156: 93-
Kehrer JP and Lund LG (1994). Cellular reducing equivalents and oxidative stress. Free Red
Biol Med. 17: 65-70.
Kidd PM (1991). Natural antioxidants Ñ first line of defense. In: Kidd PM, Huber W. Living
with the AIDS Virus: A Strategy for Long-Term Survival. Albany, California: PMK Biomedical-
Nutritional Consulting. 115-142.
Kosower NS and Kosower EM (1978). The Glutathione status of cells. Intl Rev Cytology.
Lewin S (1976).Vitamin C: Its Molecular Biology and Medical Potential. New York, NY:
Academic Press: 42-59.
Lomaestro BM and Malone M (1995). Glutathione in health and disease: Pharmacotherapeutic
issues. Annals Pharmacotherapy. 29: 1263-73
Meister A (1976). Glutathione metabolism and transport. In: Nygaard OF, Simic M G, ed.
Radioprotections and Anticarcinogens. New York, NY: Academic Press.
Quig D (1998). Cysteine metabolism and metal toxicity. Alter.Med. Rev. 3:262-270
Slater AFG, Stefan C and Nobel I (1995). Signalling mechanisms and oxidative stress in
apoptosis. Toxicol Letts; 82/83:149-153.
Stohs S J and Bagchi D ( 1993). Oxidative mechanisms in the toxicity of metal ions.Free Radic.
Biol. Med. 18: 321-336
Sidney Draggan (Topic Editor). 2007. "Health Effects Of Aluminum." In: Encyclopedia Of
Earth. Eds. Cutler J. Cleveland (Washington, D.C.: Environmental Information Coalition,
National Council For Science And The Environment).