Transcript GLUTATHIONE SYNTHESIS, EFECT OF NUTRITION ON IT …

GLUTATHIONE SYNTHESIS,
EFECT OF NUTRITION ON
REGULATORY CONTROL
MECHANISM
Mohammad Hanafi, MBBS (Syd).,dr.,MS.)
Senior Lecturer in Biochemistry
Medical Faculty Unair
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1.Introducion
• Glutathione (γ-glutamylcysteinylglycine,
GSH) is a sulfhydryl (-SH) antioxidant,
antitoxin, and enzyme cofactor.
• Glutathione can be found in animals,
plants, and microorganisms.
• It is found mainly in the cell cytosol and
other aqueous phases of the living system
• It one of the most highly concentrated
intracellular antioxidants.
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Introducion (continue)
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Introducion (continue)
• Reduced or oxidized form (GSH GSSG)
• In the healthy cell GSSG, the oxidized
(electron-poor) form, rarely exceeds 10
percent of total cell glutathione.
• Studies have led to the free radical theory
of human diseases and to the
advancement of nutritional therapies to
improve GSH status under various
pathological conditions.
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Introducion (continue)
• The adequate provision of sulfurcontaining amino acids as well as
glutamate or glutamine and glycine (or
serine) is critical for the maximization of
GSH synthesis.
• This article reviews the synthesis of
glutathione and the nutritive factors that
may modulate the synthesis of glutathione.
• Methionine and selenium requirement
described in more detail.
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2.Glutathion synthesis
• The first step of GSH synthesis is ratelimiting and catalyzed by glutamatecysteine ligase (GCL) formerly referred to
as -glutamylcysteine synthetase (GCS).
This enzyme absolutely require either Mg++ or Mn++.
• GCL, heterodimer made up of the heavy
sub unit (73 kDa) has the catalytic activity
(GCLC), and is the site of GSH feed back
inhibition
• The light (30 kDa) or modifier (GCLM) sub
unit, alter, or regulate the activity of the
holoenzyme
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Glutamate cysteine ligase (GCL)
L-glutamate + L-cysteine + ATP
-L-glutamyl-L-cysteine + ADP + Pi
GCL is regultate physiologically by :
• Feed back competitive inhibition by
GSH
• The availability of its precurser,
L-cysteine.
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The availability of its precurser,
L-cysteine.
• The cysteine content of the diet
Cysteine transport
Cystine transport
Methionine transport
Transsulfuration pathway
liver
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Cysteine transport
•
•
•
•
Na+ dependent
High stereospecificity
pH sensitive
Not sensitive to adaptive regulation or
insulin and glucagon stimulation
• Both inward and outward flows
• Influence by the extracellular levels of
cysteine, and other system amino acids
transporters
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Cystine transport
• Na+ independet
• Influence by pH change
• Entry of cystine accompanied by an exit of
glutamate
• High intracellular glutamate concentration
functions to stimulate the influx of cystine to
maintain an adequate balance between cysteine
and glutamate inside the cells
• Influence by : insulin, electrophilic agents and
O2.
• In isolated hepatocytes, treatment with
electrophilic agents depletes intracellular GSH
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Methionine transport
• Na+ independent
• Not responsive to either adaptive control
or hormonal stimulation
Cys, Met, and Cys transport
Rat hepatocytes cultured for 24 h,
the rate of uptake of cysteine is about
3-fold higher than that of methionine
and 13-fold higher than that of
cystine.
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Transsulfuration pathway
• Ability for the liver cell to convert methionine to
cysteine is important since the liver is the major
site of methionine catabolism and the major
storage organ for GSH
• It is absent or insignificant in other GSHsynthesizing systems
• Markedly impaired or absent in the fetus and
newborn infant, cirrhotic patients, and patients
with homocystinemia
• the Km of hepatic methionine
adenosyltransferase for ATP is high (2 mM)
• hypoxic depletion of ATP is more likely to affect
GSH synthesis from methionine than cysteine12
Transsulfuration pathway
• Methionine and homocysteine are readily
interconvertible
• The transsulfuration pathway converts
methionine to cysteine, which is then converted
to GSH via the GSH synthetic pathway
• Methionine can also be resynthesized from
homocysteine
• The control appears to be exerted at the level
of homocysteine: when methionine is needed,
homocysteine is remethylated by methionine
synthase or betaine-homocysteine
methyltransferase; when methionine is in
excess, catabolism of homocysteine via the
cystathionine synthase reaction is accelerated
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1.methionine adenosyltransferase
Transsulfuration pathway
Folate, vit B12
8. methionine synthase
9.betaine-homocysteine methyltransferase.
Vit B6
2.Transmethylation reaction
3.S-adenosylhomocysteine hydrolase
5.gama-cystathionase
4.cystathionine β-synthase
6,γ-glutamylcysteine synthetase
(GCL)
7.GSH synthetase
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Glutamate-cysteine ligase (GCL) activity
• Other major determinant of the rate of
GSH synthesis
• Affecting heavy (GCLC) or both the heavy
and light (GCLM)
• Transcriptional and post-transcriptional
regulation of both subunits
Post-transcriptional
-both mRNA stabilization
-destabilization and
-post-translational modification
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Activity of GCL will be influenced by :
• Oxidative stress, insulin and many others
increase GCL transcription or activity in
variety of cells
• Dietary protein deficiency,dexamethasone,
and GCL phosphorylation decrease GCS
transcription or activity
• NO production
↑ NO production
loss of GSH
↓ NO production
prevent GSH
depletion
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Glutathione synthase (GS)
-L-glutamyl-L-cysteine + glycine + ATP
GSH + ADP + Pi
• The regions of the active site that bind glycine
and the cysteinyl moiety of γ-glutamylcysteine
are highly specific
• It’s activity is modified ADP
• GSH synthase is not subject to feedback
inhibition by GSH
• GSH synthase deficiency
γ-glutamylcysteine is converted to
5-oxoproline
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Biosynthesis of glutathione
Glutamate-cysteine ligase (GCL)
GSH synthase
(GS)
-Glu-Cys-Gly
disulfide
bond
S
S
-Glu-Cys-Gly
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Glutathione distribution
• Glutathione synthesis in the body occure mainly in liver
• About 80 percent of the GSH synthesized in the liver is
exported from the hepatocytes
• Most of this is utilized by the kidneys
• GSH, to supply cysteine as needed
• Circulating GSH is safe; it reacts only slowly with
Oxygen, is less susceptible to auto-oxidative degradation
than is cysteine
• Soluble in the plasma
• GSH comes in with the diet (150 mg daily by rough
estimate)
• Hormones and other vasoactive substances increase
GSH efflux into the bile
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Strategies for Repleting Cellular Glutathione
• Oral dosing with GSH
An oral bolus of 15 mg/kg to the human
to raise plasma GSH two-to five-fold
The intestinal lumen absorb GSH via non-energyrequiring, carrier-mediated diffusion, and later export
it into the blood
(lung alveolar cells, vessel endothelial cells, retinal
pigmented epithelial cells, and cells of the kidney's
proximal tubule;
it seems also to cross the blood-brain barrier;
while brain endothelial and nerve cells, red blood
cells, lymphocytes - appear incapable of absorbing
GSH as the intact tripeptide )
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•
•
•
•
L-methionine
Is an essential amino acid
It must first be converted to cysteine
This convertion is inactive in neonates and
in certain adults, such as patients with liver
disease
The "activated" methionine metabolite
known as SAM (S-adenosyl methionine) is
effective in raising red cell GSH and
hepatic GSH when given orally at 1600 mg
per day
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•
•
•
•
L-methionine
L-methionine intake is 13 mg per kg or
about 0.75 gram daily for adults
During methionine supplementation, intake
B6 and folic acid should also be included
Excessive methionine intake, together with
inadequate intake of folic acid, vitamin B6,
and vitamin B12, can increase the
conversion of methionine to homocysteine
Homocysteine is a potentially harmful
blood fat that has been linked to
atherosclerosis
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Table 1. Methionine content of 100gr edible food in mg.
No. Description
Weight (mg)
1.
Beef loin
540
2.
Beef liver
410
3.
Lamb
360
4.
Egg (whole)
1000
There are about 15 to 16 eggs in one kg
( at Rungkut market),
ie equal to 66.7 - 62.5 gr/egg
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L-cysteine
• Cysteine is probably unsafe for routine
oral administration
• In the blood it readily auto-oxidizes to
potentially toxic degradation products
• The auto-oxidation
Hydroxyl radical
• The cystine produced from cysteine
oxidation is taken up into the kidney, and
requires energy and enzymatic
intervention to be converted to cysteine
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N-acetyl cysteine (NAC)
• NAC is a cysteine precursor; it is well absorbed by the
intestine
• Metabolized by the liver, cysteine is one of metabolite
• It seems not to raise GSH levels if they are already
within the normal range
• It can raise abnormally low GSH levels back to normal
• This is the basis for its use as an antidote to
acetaminophen's liver toxicity
• Used in Europe for many years as a mucolytic agent
• 600 mg was beneficial and innocuous while 1200 mg
and 1800 mg per day caused significant adverse effects
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Alpha lipoic acid
• Alpha lipoic acid (ALA) is available as
supplement
• It is a natural antioxidants and could
increase intracellular glutathione through
it reducing power
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Selenium
• Is a co-factor for the enzyme glutathione
peroxidase
• Studies suggest they may play a role in
decreasing the risk of certain condition in which
glutathione are depleted
• Too much selenium can cause toxic effects
including gastrointestinal upset, brittle nails, hair
lost and mild nerve damage
• Upper estimated requirement of 90 μg Se/d
• Lower estimated requirement of 39 μg Se/d
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Table 2. Selenium content of food in μg unit
No.
Description
Weight
(gram)
Content per
measure (μg )
1.
2.
3.
Asparagus 4 spears
Beef, sirloin
Bread white 1 slice
60
85
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3.7
24.8
0.8
4.
5.
6.
7.
Chicken breast
Duck
Egg fried
Lamb
98
221
46
85
23.7
49.5
15.7
27.9
8.
9.
Rice 1 cup cook
Tuna
175
85
14.3
55.8
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Concluding remark
• Advances in molecular biology have led to an
explosion of knowledge in understanding how
the rate-limiting enzyme GCS is regulated at the
molecular level, and prevention of complications
that may result from altered GSH synthesis
• Protein (or amino acid) deficiency remains a
significant nutritional problem in the world to
day. In developing country where source of
protein is very expensive beyond their budget
owing to inadequate income; under nutrition,
specially protein deficiency become a great
problem
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Concluding remark (continue)
• Thus, new knowledge regarding the efficient utilization of
dietary protein or the precursors for GSH synthesis
and its nutritional status is critical for the development
of effective therapeutic strategies to prevent and treat a
wide array of human diseases, especially those condition
or diseases that are related to oxidative stress
• High methionine content of meat, fish, and dairy product
probably meet the daily requirement. In certain condition
such as hepatic cirrhosis the ability to convert
methionine to SAM is disturbed. Therefore methionine is
given in SAM form. SAM is easily available as drug in
Europe
• Selenium is cofactor for glutathione peroxidase.
Suffice to take an action by consuming it even though
research still needed to confirm the cost benefits. Food
consumed every day contain selenium, probably
enough to meet our need
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