GLUTATHIONE SYNTHESIS, EFECT OF NUTRITION ON REGULATORY CONTROL MECHANISM

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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 (?-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 sulfur-containing 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 rate-limiting
  and catalyzed by glutamate-cysteine 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 liver
• Cysteine transport
• Cystine transport
• Methionine transport
• Transsulfuration pathway

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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
  GSH-synthesizing 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 cysteine

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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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Transsulfuration pathway
1.methionine adenosyltransferase
Folate, vit B12
8. methionine synthase
9.betaine-homocysteine methyltransferase.
Vit B6
2.Transmethylation reaction
3.S-adenosylhomocysteine hydrolase
5.gama-cystathionase
4.cystathionine ß-synthase
6,?-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 ?-glutamylcysteine
  are highly specific
• Its activity is modified ADP
• GSH synthase is not subject to feedback
  inhibition by GSH
• GSH synthase deficiency
• ?-glutamylcysteine is converted to
• 5-oxoproline

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Biosynthesis of glutathione
GSH synthase (GS)
Glutamate-cysteine ligase (GCL)
disulfide bond
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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-energy-
• requiring, 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. Asparagus 4 spears 60 3.7
2. Beef, sirloin 85 24.8
3. Bread white 1 slice 22 0.8
4. Chicken breast 98 23.7
5. Duck 221 49.5
6. Egg fried 46 15.7
7. Lamb 85 27.9
8. Rice 1 cup cook 175 14.3
9. Tuna 85 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