S' Jill James, Ph'D'

1
Pathogenic Implications of Low Glutathione
Levels And Oxidative Stress in Children with
Autism Metabolic Biomarkers and Genetic
Predisposition
S. Jill James, Ph.D. Department of
Pediatrics Arkansas Childrens Hospital Research
Institute University of Arkansas for Medical
Sciences Little Rock, AR
2
Overview
A little Basic Biochemistry Folate/Methionine/Glu
tathione What is Glutathione and Why is it
Important? What is Oxidative Stress and How Does
it Damage Cells? Thimerosal Toxicity and
Glutathione Depletion In Vitro Oxidative Stress
and reduced methylation capacity in Autistic
Children Results of an Intervention Trial with
Methyl B-12, Folinic Acid, and TMG
Increased Frequency of Selected Genetic
Polymorphisms Associated with the Abnormal
Metabolic Profile in Autism Implications of
Oxidative Stress in the Pathogenesis of Autism
3
Methionine Transsulfuration to Cysteine and
Glutathione
Methionine
THF
1
5,10-CH2THF
MS
B12
MTHFR
5-CH3THF
Homocysteine
B6
THF tetrahydrofolate
Enzymes
4
Methionine Transsulfuration to Cysteine and
Glutathione
Methionine
THF
1
5,10-CH2THF
MS
B12
MTHFR
5-CH3THF
Homocysteine
B6
THF tetrahydrofolate
Enzymes
5
Methionine Transsulfuration to Cysteine and
Glutathione
Methionine
Methylation Potential (SAM/SAH)
THF
SAM
MTase
1
5,10-CH2THF
Cell Methylation
2
BHMT
MS
SAH
B12
MTHFR
Betaine
5-CH3THF
SAHH
Choline
Adenosine
Homocysteine
B6
THF tetrahydrofolate
Enzymes
6
Methionine Transsulfuration to Cysteine and
Glutathione
Methionine
Methylation Potential (SAM/SAH)
THF
SAM
MTase
1
5,10-CH2THF
Cell Methylation
2
BHMT
MS
SAH
B12
MTHFR
Betaine
5-CH3THF
SAHH
Choline
Adenosine
Homocysteine
CBS
B6
B6
Cystathionine
3
Antioxidant Potential (GSH/GSSG)
B6
THF tetrahydrofolate
Cysteine
Enzymes
GSH GSSG
7
Methionine Transsulfuration to Cysteine and
Glutathione
Methionine
Methylation Potential (SAM/SAH)
THF
SAM
MTase
1
5,10-CH2THF
Cell Methylation
2
BHMT
MS
SAH
B12
MTHFR
Betaine
5-CH3THF
SAHH
Adenosine
Choline
Homocysteine
1
Folate Cycle Methionine Cycle Transsulfuration
Pathway
CBS
B6
B6
Cystathionine
2
3
Antioxidant Potential (GSH/GSSG)
B6
Cysteine
3
GSH GSSG
8
RELEVANT BACKGROUND INFORMATION
Methionine is an essential AA Methionine Cycle
conserves methionine Cysteine is a
conditionally essential AA gt50 of cysteine
is derived from methionine via the
transsulfuration pathway normal cysteine levels
depend on normal methionine levels. Cysteine
may be an essential amino acid in children with
autism! Cysteine is the rate-limiting amino acid
for glutathione (Glu-Gly-Cys) synthesis the
thiol (-SH) group of cysteine is the active
antioxidant component of glutathione. Females
have higher methionine cycle turnover and higher
GSH levels than males Higher methylation and
antioxidant capacity in girls could be
protective and could contribute to the skewed
gender ratio in autism
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How Does Oxidative Stress Influence
Methionine Metabolism?
10
Impact of Oxidative Stress on Methionine
Transsulfuration
Methionine
Protein synthesis
THF
SAM
Methylation of DNA, RNA, Proteins,
Catecholamines, Phospholipids, Creatine
MTase
MS
BHMT
SAH
B12
Betaine
AK
SAHH
Choline
AMP
Adenosine
5-CH3THF
ADA
Homocysteine
Inosine
CBS
B6
B6
THF tetrahydrofolate
Cystathionine
Transsulfuration Pathway
Cysteine
Enzymes
Glutathione
11
SOURCES OF OXYGEN FREE RADICALS AND CELL INJURY
Oxidative Stress
12
MECHANISMS OF FREE RADICAL-MEDIATED CELLULAR
INJURY
13
FREE RADICAL CELLULAR DEFENSE MECHANISMS
Glutathione
Glutathione
Glutathione
Glutathione
Glutathione
Cytoplasm
Glutathione
Cytoplasm
Glutathione
Glutathione
Glutathione
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ANTIOXIDANT DEFENSE COUNTERBALANCES OXIDATIVE
STRESS
Oxidative Stress Antioxidant Defense
Superoxide Dismutase GSH Peroxidase GSH
Reductase Vitamin E Vitamin C Lipoic
Acid GSTs GSH
Hydroxyl Radical Hydrogen Peroxide Superoxide ONOO
- GSSG 4HNE LOO- NO-

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Oxidative Stress Antioxidant Defense
Superoxide Dismutase GSH Peroxidase GSH
Reductase Vitamin E Vitamin C Lipoic
Acid GSTs GSH

Hydroxyl Radical Hydrogen Peroxide Superoxide ONOO
- GSSG 4HNE LOO-
Cell Death Damage
16
ANTIOXIDANT FUNCTION OF GLUTATHIONE
Major intracellular antioxidant H2O2,
superoxide, hydroxyl radical,
peroxynitrite, membrane lipid peroxidation
(active) (inactive)
17
DETOXIFICATION FUNCTIONS OF GLUTATHIONE
Maternal/Fetal Drug/Carcinogen Exposures
GST
Heavy Metals
Glutathione Conjugate
Glutamine
Glycine
Mercapturic Acid (Cysteine conjugate)
Bile and Urine Excretion
Cysteine loss increased requirement for de
novo glutathione synthesis sulfur loss in urine
Detoxification Hg, As, Pb, Cd bind to thiol
(SH) group Metal-cysteine conjugates excreted
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Functional Consequences of Low GSH/GSSG and
Increased Oxidative Stress
Reduced ability to detoxify environmental
toxicants and heavy metals Neurotoxicity
Immunotoxicity Oxidation of active site cysteine
(-SH groups) in enzymes altered
structure/function Abnormal methionine
metabolism Altered membrane signaling
Decreased liver GSH synthesis reduced export
of cysteine to brain Reduced
astroglial/neuronal GSH synthesis increased
sensitivity to heavy metals Neurotoxicity
Integrity of the gut epithelium compromised
Increased mucosal membrane permeability
malabsorption Altered T-cell subpopulations
Decreased Th1Increased Th2 autoimmunity
gut inflammation
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Neurotoxicity of Thimerosal in Human Brain
Cells is Associated with Glutathione Depletion
Protective Effect of N-Acetyl Cysteine or
Glutathione
S. Jill James, William Slikker, Elizabeth New,
Stefanie Jernigan, Stepan Melnyk
Neurotoxicology, volume 26 pp1-8, 2005
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WORKING HYPOTHESIS
The neurotoxicity of Thimerosal is associated
with depletion of glutathione, the major
intracellular antioxidant Ethyl mercury in
Thimerosal binds to cysteine thiol (SH)
groups on intracellular proteins and inactivates
function. The cysteine-SH group of
glutathione, binds mercury and protects
essential proteins from functional inactivation.
Glutathione is the major mechanism of mercury
detoxification
21
Intracellular glutathione levels in cells exposed
to 15 ?M Thimerosal (T) in presence of 100 ?M
N-acetylcysteine (NAC) or glutathione ethyl ester
(GSH)
GLIOBLASTOMA NEUROBLASTOMA
Intracellular GSH (nmol/mg protein)
Control T TNAC TGSH
Control T TNAC TGSH
22
THE IMPORTANCE OF AGE AT EXPOSURE TO THIMEROSAL
INSULT

• Infants and children are not tiny adults higher
  metabolic rate, higher respiratory rate, rapid
  cell proliferation, smaller volume i.e., an
  equivalent dose will be more toxic to a child
• The transsulfuration pathway to glutathione is
  immature in the fetus and infant less able to
  handle pro-oxidant exposures
• Simultaneous exposure to Thimerosal and other
  heavy metals is additive in the developing brain.

4. Children with a genetic predisposition to
impaired antioxidant defense will reach
the critical threshold for developmental
damage earlier than those not genetically at
risk.
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Increased Oxidative Stress and Impaired
Methylation Capacity in Children with Autism
Metabolic Biomarkers and Genetic
Predisposition Results of Intervention Trial
with Folinic Acid, TMG, and Methyl-B12
S. Jill James, Ph.D., Laurette Janak, M.O.M.,
Stepan Melnyk, Ph.D., Stefanie Jernigan, Paul
Cutler, M.D.
American Journal of Clinical Nutrition volume 80
pp 611-17,2004
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Folinic Acid, TMG, and Methyl B-12
Supplementation in Children with Autism
Phase 1 Baseline levels of metabolites were
measured in 20 children with autism and compared
with 33 control children. Phase 2 Eight of
the children participated in an
intervention trial and were given 800 µg folinic
acid and 1000 mg betaine b.i.d. for 3 months and
the plasma metabolites were re-measured. Phase
3 The children were then given injectible
methyl-B12 (75 µg/Kg 2x/week) and the plasma
profile was repeated after 4 weeks of combined
folinic acid, betaine, and methyl B12
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Methionine Cycle Metabolites
Control Children Autistic
Children p value n33
n20
Methionine (µmol/L) 31.5 ? 5.7 19.3 ?
9.7 0.001 SAM (nmol/L) 96.9 ? 12
75.8 ? 16.2 0.01 SAH
(nmol/L) 19.4 ? 3.4 28.9 ? 7.2
0.001 SAM/SAH ratio 5.2 ? 1.3
2.9 ? 0.8 0.001 Adenosine (µmol/L) 0.27 ?
0.1 0.39 ? 0.2
0.05 Homocysteine (µmol/L) 6.4 ? 1.3
5.8 ? 1.0 0.01
26
INTERPRETATION

• The decrease in methionine and homocysteine
  levels in autistic
• children indicates that they have reduced
  methionine synthase
• activity and reduced turnover of the
  methionine cycle.
• We need to jump start this pathway!
• The decrease in SAM and increase in SAH provides
  metabolic evidence
• that methylation capacity is reduced in some
  autistic children.
• We need to normalize the SAM/SAH ratio
• and normalize methylation capacity!
• 3. The increase in SAH is most likely due to
  the increase in adenosine
• that inactivates SAH hydrolase activity
• We need to reduce adenosine levels!

27
Transsulfuration Metabolites
Control Children Autistic Children
p value n33
n20
Homocysteine (µmol/L) 6.4 ? 1.3 5.8
? 1.0 0.01 Cystathionine (µmol/L) 0.17
? 0.05 0.14 ? 0.06 0.002 Cysteine
(µmol/L) 202 ? 17 163 ? 15
0.001 Total glutathione (µmol/L) 7.6 ? 1.4
4.1 ? 0.5 0.001 Oxidized
Glutathione (nmol/L) 0.32 ? 0.1 0.55 ?
0.2 0.001 GSH/GSSG Ratio 25.5 ?
8.9 8.6 ? 3.5 0.001
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INTERPRETATION

• The significant decreases in homocysteine,
  cystathionine, cysteine and glutathione indicate
  that the transsulfuration pathway is depressed in
  autistic children.
• The decrease in these metabolites is consistent
  with the
• decrease in methionine levels.
• We need to get methionine back up to jump start
  this pathway!
• The increase in GSSG (oxidized inactive
  glutathione) and decrease in GSH (active
  antioxidant glutathione) is strong evidence that
  oxidative stress is increased in autistic
  children.
• We need to normalize GSH/GSSG ratio
• and reduce oxidative damage!

29
How can we feed this pathway to strengthen
SAM/SAH and GSH/GSSG potential?
30
DNA synthesis
Methionine
Protein synthesis
dNTPs
THF
SAM
DMG
MTase
Methylation of DNA, RNA, histones, membrane
phospholipids
BHMT
MS
5,10 CH2 THF
SAH
Me-B12
Betaine
Folinic Acid
SAHH
Adenosine
5CH3THF
Homocysteine
CBS
Supplementation 800 µg folinic acid, b.i.d. 1000
mg betaine, b.i.d. 75 µg/Kg methyl-B12
B6
Cystathionine
Transsulfuration Pathway
Cysteine
Glutathione
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How Does Methyl B12 Work and Why does
Folinic Acid Help?
32
B12
CH3
Homocysteine
MTRR
Folate
Oxidized B12
CH3 Methyl Group
Methionine Synthase
33
Metabolically Active Folate
CH3
B12
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
34
Metabolically Active Folate
Methionine
B12
CH3
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
35
B12
CH3
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
36
Metabolically Active Folate
CH3
B12
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
37
Metabolically Active Folate
Methionine
B12
CH3
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
38
Low Glutathione and Oxidative Stress
Metabolically Active Folate
B12
CH3
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
39
Low Glutathione and Oxidative Stress
CH3-B12
Metabolically Active Folate
CH3-B12
CH3
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
40
Low Glutathione and Oxidative Stress
CH3-B12
Metabolically Active Folate
Methionine
CH3-B12
CH3
CH3
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
41
Low Glutathione and Oxidative Stress
CH3-B12
Metabolically Active Folate
Methionine
CH3-B12
Betaine
Folinic Acid
CH3
CH3
Homocysteine
MTRR
Folate
Oxidized B12
Methionine Synthase
42
Proportion of Autistic Children within Normal
Range Before and After Supplementation

FolinicBetaine Metabolite
Normal Range a Baseline FolinicBetaine
methylB12 Methionine (?mol/L) gt 24
1/8 5/8 7/8 SAM (nmol/L)
gt 80 2/8 8/8 8/8
SAH (nmol/L) lt 23 2/8
7/8 7/8 SAM/SAH gt 4
1/8 7/8 7/8 Adenosine (?mol/L)
lt 0.3 4/8 8/8
8/8 Homocysteine (?mol/L) lt 5.5
3/8 8/8 8/8 Cysteine
(?mol/L) lt180 0/8
2/8 7/8 GSH (?mol/L)
gt 5.4 0/8 2/8
7/8 GSSG (?mol/L) lt 0.33
0/8 2/8 8/8
GSH/GSSG gt 16 0/8
3/8 8/8 __________________________
__________________________________________________
a Range estimated to include 90 of control
children
43
INTERPRETATION
Folinic Acid and Betaine brought all the
methionine cycle metabolites into normal
range. The combined regimen of Folinic Acid,
Betaine, and Methyl B12 brought all the
transsulfuration metabolites into the normal
range. Best predictors of impaired methylation
are low methionine and SAM/SAH ratio OR elevated
adenosine. Best predictors of impaired
antioxidant defense are low cysteine, and low
glutathione (low GSH/GSSG ratio).
44
Expanded Baseline Data and Lessons Learned
We now know after analyzing baseline
metabolites from over 100 autistic
children 1. Each child is unique one size
(dose) does not fit all 2. Low cysteine, low
free glutathione, low GSH/GSSG Ratio
(reduced antioxidant capacity) are present in
over 80 of autistic children. 3.
Increased SAH and elevated adenosine (reduced
methylation capacity) only occurs in a subset of
about 21 of autistic children. 4. About
20 of children with autism do not tolerate
TMG (become hyperactive) and about 5 do not
tolerate folinic acid or methylB12.
45
Important Caveat
The abnormal metabolic profile reflects the
presence of autism but we do not yet know if it
is predictive of the risk of autism (e.g.,
whether profile is a cause or consequence of
autism).
46
Is there a genetic basis for increased
vulnerability to oxidative stress? e.g.,
Mercury and other heavy metal toxicity cell
death Autoimmunity increased T helper2
cells Gut Inflammation increased
inflammatory cytokines Redox imbalance
in the brain inflammation cell death
Redox enzyme inhibition excess dopamine,
glutamate
47
SPECIFIC GENETIC CODE FOR MTHFR
C
A
G
T
48
GENETIC POLYMORPHISM IN MTHFR AT POSITION 677
MTHFR 677 C?T
C?T
A
G
T
RESULTS IN AN ABNORMAL PROTEIN WITH REDUCED
ENZYME ACTIVITY
49
GENETIC POLYMORPHISM IN MTHFR AT POSITION 677
MTHFR 677 C?T
C?T
A
G
T
TEXT ANALOGY COT CAT
50
Metabolic Response to Genetic Polymorphisms in
the Methionine Cycle
Methionine
THF
SAM
Methyl Acceptor
DMG
Methyltransferase
5,10-CH2-THF
B12
COMT
TC II
Methylated Product
MTHFR
5-CH3-THF
SAH
RFC
Adenosine
Homocysteine
Cystathionine
Cysteine
Glutathione
GST
51
Functions of Polymorphic Genes Analyzed
MTHFR Methylenetetrahydrofolate reductase
Transfers methyl groups for methionine
synthesis MTRR Methionine synthase reductase
May reduce methionine synthase activity
RFC Reduced Folate CarrierTransports
folate into the cell GSTs Glutathione-S-
Transferase Important for glutathione
detoxification capacity TCII Transcobalamin
II Transports methylB12 into the cell COMT
Catechol-O-methyltransferase Methylates
dopamine and prevents dopamine-induced
oxidative stress in the brain APO E4 May help
detoxify mercury
52
Polymorphisms in the Methionine Cycle Pathway
Methylenetetrahydrofolate Reductase
(677C?T1298A?C)
1. MTHFR 677 TT
Frequency Odds Ratio p value
Control Individuals (203) 10.7
Autistic Children (360) 13.1
1.47 NS 2. MTHFR 677
CT Control Individuals (205)
43.9 Autistic Children (360)
49.2 1.35
NS 3. MTHFR 677CT/1298AC Control
Individuals (205) 19.1
Autistic Children (360) 23.7
1.7 NS 0.07
NS Not statistically significant
These results do not repeat earlier observations
53
Polymorphisms in the Methionine Cycle Pathway
Methionine Synthase Reductase (MTRR 66A?G)
1. MTRR 66 GG
Frequency Odds Ratio p value
Control Individuals (203) 33.2
Autistic Children (360)
28.0 0.61 0.06 2. MTRR 66 AG
Control Individuals (205)
48.5 Autistic Children (360)
46.7 1.4
NS
MTRR G allele is less frequent in autistic
children
54
Polymorphisms in the Methionine Cycle Pathway
Transcobalamin II (TCII 776 66C?G)
1. TCII 776 GG
Frequency Odds Ratio p value
Control Individuals (203) 16.0
Autistic Children (360)
25.8 1.8 0.05 2. TCII 776
CCCG Control Individuals (205)
84.0 Autistic Children
(360) 74.2 0.55
0.01
Statistically significant
TCII G allele is significantly more frequent in
autistic children Theoretically, this could
reduce B12 transport inside the cell and could
decrease methionine synthase activity
55
Polymorphisms in the Methionine Cycle Pathway
Reduced Folate Carrier RFC1 80A?G
RFC1 80 GG Frequency
Odds Ratio 95 C.I. Control
Individuals (203) 30 Autistic
Children (360) 39 2.0
1.15,3.33
56
Polymorphisms Glutathione-S-Transferase
(GST) Affecting Antioxidant
Capacity
GST M1 and GST T1
1. GST M1 Null
Frequency Odds Ratio p
value Control Individuals (205) 57.2
Autistic Children (360) 49.2
1.4 0.07, NS 2. GST T1 Null
Control Individuals (183)
25.8 Autistic Children (233) 22.7
0.86 NS
57
Polymorphisms Affecting Methylation and
Increased Oxidative Stress
Catechol-O-Methyltransferase (COMT 1947A?G)
COMT 1947GG (low activity variant)
Frequency Odds Ratio 95 C.I. Control
Individuals (205) 16.3 Autistic Children
(360) 26 2.34 1.06,2.85

58
Polymorphisms Affecting Methylation and
Increased Oxidative Stress
APOPROTEIN E4 (APO E4)
APO E4 (E3/E4 or E4/E4) Control
Individuals (205) 25 Autistic
Children (360) 27 0.96
NS
Frequency Odds Ratio p value
59
Gene-Gene Interactions Affecting
Methylation and Increased Oxidative Stress
Combined TCII GG plus COMT GG in the same
individual
TCII GG/COMT GG Frequency Odds Ratio
95 C.I. Control Individuals (203)
2.5 Autistic Children (360)
9.7 7.0 2.32,21.2

The TCII and COMT gene polymorphisms may interact
to synergistically increase the risk of autism
60
Gene-Gene Interactions Affecting
Methylation and Increased Oxidative Stress
Combined TCII GG plus GSTM1 null in the same
individual
TCII GG/GSTM1 null Frequency Odds Ratio
95 C.I. Control Individuals (203)
7.6 Autistic Children (360)
13.4 2.2 1.05,4.4

The TC II and GSTM1 null gene polymorphisms
interact to increase the risk of autism
61
Gene-Gene Interactions Affecting
Methylation and Increased Oxidative Stress
Combined RFC1 GG plus COMT AG in same individual
RFC1 GG/COMT GG Frequency Odds Ratio
95 C.I. Control Individuals (203)
6.7 Autistic Children (360)
13.8 2.5 1.05,5.75

Polymorphisms in the RFC1 and COMT genes interact
to increase risk of autism
62
Important Caveat
No single polymorphism alone can predict
increased risk of autism because, by definition,
polymorphisms are highly prevalent in normal
people as well. It is possible, however, that
specific combinations of these polymorphisms
interact to shift specific metabolic pathways
that are important in the pathogenesis of
autism.
To determine if a relationship exists between
metabolic profile and genetic profile will
require statistical analysis of 1500 cases and
1500 control children. Were not there yet
but the NIH grant is in!!
63
PUTTING IT ALL TOGETHER.....
64
Metabolic Indicators
Reduced Methionine (Oxidative inhibition of MS)
Reduced SAM (Low methionine) Reduced
Cysteine (Low methionine, Homocysteine) Reduced
Glutathione (Low Cysteine)
Reduced Cellular Antioxidant Capacity in Autism
65
Factors Contributing to Oxidative Stress
in Autistic Children
Inflammation Infection
Autism
Genes
Environment
Hormones
Gut Inflammation Brain Inflammation Immune
dysfunction
Timing
66
Putting it all into Perspectivewe see what we
know
Cellular Metabolic Pathways
You Are Here
67
OVERALL CONCLUSION NOVEMBER 2005
The abnormal metabolic profile in children with
autism strengthens the hypothesis that a genetic
inability to control oxidative stress may be
central to the development of neurologic,
immunologic, and gastrointestinal dysfunction
that occurs with autism.
68
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