Evidence for Increased Oxidative Stress and Impaired

1
Evidence for Increased Oxidative Stress and
Impaired Methylation 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? Abnormal Methylation and
Oxidative Stress 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 and
Methylation Deficit in the Pathogenesis of Autism
3
Folate-Dependent Methionine Cycle
Methionine
Protein synthesis
THF
SAM
Methylation of DNA, RNA, Proteins,
Catecholamines, Phospholipids, Creatine
MTase
MS
SAH
B12
AK
SAHH
AMP
Adenosine
5-CH3THF
ADA
Homocysteine
Inosine
THF tetrahydrofolate
Enzymes
4
Folate-Dependent Methionine Cycle
Methionine
Protein synthesis
THF
SAM
Methylation of DNA, RNA, Proteins,
Catecholamines, Phospholipids, Creatine
MTase
MS
SAH
B12
AK
SAHH
AMP
Adenosine
5-CH3THF
ADA
Homocysteine
Inosine
THF tetrahydrofolate
Enzymes
5
Folate-Dependent Methionine Cycle
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
B6
THF tetrahydrofolate
Enzymes
6
Methionine Transsulfuration to Cysteine and
Glutathione
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
B6
Transsulfuration Pathway
Cysteine
Enzymes
Glutathione
7
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
8
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
9
SOURCES OF OXYGEN FREE RADICALS AND CELL INJURY
Oxidative Stress
10
MECHANISMS OF FREE RADICAL-MEDIATED CELLULAR
INJURY
11
FREE RADICAL CELLULAR DEFENSE MECHANISMS
Glutathione
Glutathione
Glutathione
Glutathione
Glutathione
Cytoplasm
Glutathione
Cytoplasm
Glutathione
Glutathione
Glutathione
12
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-

13
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
14
ANTIOXIDANT FUNCTION OF GLUTATHIONE
(active) (inactive)
15
GLUTATHIONE AS FREE RADICAL SCAVENGER
Aerobic Metabolism (ATP synthesis in
mitochondria)
.
GSH
O2
OH.
Fe
SOD
H2O2
Peroxisomes
NADP
2 GSH
GpX GSSG-Reductase
GS-SG
NADPH
H2O
Major intracellular antioxidant H2O2,
superoxide, hydroxyl radical,
peroxynitrite, membrane lipid peroxidation
16
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
17
REGENERATION OF GLUTATHIONE IMPORTANCE FOR
TOTAL ANTIOXIDANT CAPACITY
2 GSH
?-Tocopherol
NADP
Dehydro Ascorbic Acid
(Active)
(Inactive)
(Active)
(Inactive)
Oxidized ?-Tocopherol
Ascorbic Acid
NADPH
GSSG
(Active)
(Inactive)
(Inactive)
(Active)
18
Additional Cellular Functions of Glutathione
Major Route of Mercury Excretion
Glutathione/cysteine conjugates excreted in
urine and in bile Protein redox status
Prevents oxidation of cysteine residues in
proteins and functional inactivation of
proteins ATP production and mitochondrial
integrity Protects mitochondrial membrane
integrity Integrity of gut epithelium GSH
depletion associated with degeneration
mucosal villi and increased gut
permiability Normal T cell subsets and immune
function Normal T cell maturation in
the thymus
19
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
Recently accepted to Neurotoxicology for
publication this fall!!
20
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
POTENTIAL IMPLICATIONS
The protective effect of GSH ester and NAC
against mercury toxicity suggests these nutrient
precursors could be used as adjunct therapy to
individuals receiving thimerosal-containing
vaccinations. Individuals with nutritional or
genetic deficiencies in glutathione synthesis
will be less able to excrete mercury/heavy metals
and will be more sensitive to pro-oxidant
exposures such as mercury, arsenic, cadmium, and
lead.
23
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.
24
Impaired Methylation Capacity and Increased
Oxidative Stress in Children with Autism
Metabolic Biomarkers and Genetic
Predisposition Results of Intervention Trial
with Folinic Acid, Betaine, and Methyl-B12
S. Jill James, Ph.D., Laurette Janak, M.O.M.,
Stepan Melnyk, Ph.D., Stefanie Jernigan, Paul
Cutler, M.D.
Accepted for Publication in American Journal of
Clinical Nutrition!!
25
Folinic Acid, Betaine, 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
26
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
27
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!

28
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
29
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!

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
31
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
Methionine Cycle Metabolites Before and After
Supplementation
Folinic Betaine
Baseline Folinic Betaine
B12 Methionine (µmol/L) 19.2 3.5
(low) 25.7 3.6 30.9 7.7
SAM (nmol/L) 75.5 5.0 (low) 112.9
20.8 101.6 20.5 SAH (nmol/L) 27.6
6.1 (high) 16.9 6.5 14.3 7.5
SAM/SAH ratio 2.9 0.8 (low)
7.4 4.1 8.9 4.5 Adenosine
(µmol/L) 0.30 0.2 (high) 0.18 0.04
0.14 0.03 Homocysteine (µmol/L)
5.4 0.9 (low) 6.7 0.7
7.4 1.7
43
Transsulfuration Metabolites Before and After
Supplementation
Folinic Betaine
Baseline Folinic
Betaine B12
Homocysteine (µmol/L) 5.4 0.9 6.7
0.7 7.4 1.7 Cystathionine
(µmol/L) 0.10 0.02 0.22 0.08
0.25 0.08 Cysteine (µmol/L) 166
11.4 180 11 199.3 15
Glutathione (µmol/L) 4.0 0.7
5.0 0.9 6.7 1.6 Oxidized GSSG
(nmol/L) 0.59 0.2 0.38 0.2 0.24
0.5 GSH/GSSG Ratio 7.5 2.3 13.8
4.8 28.7 7.1
44
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
45
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. Bless Jim Neubrander! 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).
46
Expanded Baseline Data and Lessons Learned

• We now know after analyzing metabolites from over
  90 autistic children
• 1. Low methionine, low cysteine, low glutathione,
  low GSH/GSSG Ratio
• are present in over 80 of autistic children
• Low SAM and SAM/SAH ratio and elevated Adenosine
  and SAH
• occur in a subset of about 21 of children
  with autism
• 3. About 30 of children with autism react to TMG
  with hyperactivity
• (need to lower dose or omit TMG if
  hyperactivity is sustained)

Interventions to Increase Glutathione Precursors
and Reduce Oxidative Stress

• To increase Methionine TMG (if tolerated) and
  Methyl-B12 Zinc
• 2. To increase active Folate and Purine
  Synthesis Folinic Acid, DMG
• To increase Cysteine and Glutathione Methyl-B12
  NAC
• Cofactors B6 and selenium
• 4. To support glutathione functions Vitamin E,
  C, and alpha lipoic acid
• 5. To support low ATP production with oxidative
  stress creatine
• 6. To reduce lipid peroxidation with oxidative
  stress DHA, EPA

47
Metabolic Response to Genetic Polymorphisms in
the Methionine Cycle
Methionine
THF
SAM
Methyl Acceptor
DMG
Methyltransferase
TC II
5,10-CH2-THF
B12
COMT
Methylated Product
MTHFR
5-CH3-THF
SAH
Adenosine
Homocysteine
Cystathionine
Cysteine
Glutathione
GST
48
Polymorphisms in the Methionine Cycle
Pathway Methylenetetrahydrofolate Reductase
(677C?T1298A?C)
Frequency Odds Ratio p
value 1. MTHFR 677 TT Control Individuals
(183) 10.9 Autistic
Children (231) 13.4 1.26
0.28 2. MTHFR 677 CT Control
Individuals (183) 44.5
Autistic Children (231) 52.8
1.4 0.05 3. MTHFR 677CT/1298AC
Control Individuals (183) 18.1
Autistic Children (231) 26.4
1.6 0.03 4. MTHFR T Allele
Frequency Control Individuals (183)
33 Autistic Children (231)
40 1.33 0.03
49
Polymorphisms Glutathione-S-Transferase
Affecting Antioxidant Capacity
1. GST M1 Null
Frequency Odds Ratio p
value Control Individuals (183)
41.5 Autistic Children (233) 50.6
1.44 0.04 2. GST T1 Null
Control Individuals (183)
25.8 Autistic Children (233) 22.7
0.95 0.42 3. GST M1/T1 Double Null
Control Individuals (183)
8.2 Autistic Children (233)
13.3 1.72 0.06
50
Polymorphisms Affecting Methylation and
Increased Oxidative Stress
1. Transcobalamin II Frequency
Odds Ratio p value TCII 776GG
Control Individuals (117) 13.6
Autistic Children (178) 26.8 2.53
0.002 2. Catecholamine-O-Methyltransferase
COMT 1947GG Control Individuals
(183) 16.3 Autistic Children
(216) 26 2.34 0.004 3.
Apoprotein E4 (APO E4) (E3/E4 or
E4/E4) Control Individuals (151)
25 Autistic Children (216)
27 0.96 0.48
51
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 pathogeneisis of
autism.
To determine if a relationship exists between
metabolic profile and genetic profile will
require statistical analysis of 800-1000
children were not there yet!
52
PUTTING IT ALL TOGETHER.....
53
Metabolic Indicators
Reduced Methionine (Oxidative inhibition of MS
MTHFR /TC II polymorphisms) Reduced SAM (Low
methionine) Increased Adenosine (ADA
polymorphism oxidative stress) Increased SAH
(SAHH inhibition by adenosine binding) Product
inhibition of cellular methyltransferases by
SAH Reduced Cellular Methylation Capacity in
Autism
54
Functional consequences of reduced methylation
capacity in children with autism
Reduced DNA methylation and silencing of
integrated viral sequences Implication for
persistent measles virus expression after
MMR? Reduced creatine synthesis (methylation of
guanidinoacetate) Reduced phosphocreatine and
ATP availability in muscle and brain Reduced
membrane phosphatidylcholine synthesis
(methylation of phosphatidylethanolamine)
Altered membrane fluidity, permeability, and
transmembrane signaling Reduced neurotransmittor
methylation and function Reduced methylation of
dopamine (elevated dopamine)
55
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
56
Functional consequences of low glutathione and
reduced antioxidant capacity in children with
autism
Reduced ability to detoxify environmental
toxicants and heavy metals Neurotoxicity
Immunotoxicity Oxidation of cysteine thiol (SH)
groups in proteins altered structure/function
Altered membrane signaling decreased tubulin
formation Decreased liver GSH synthesis
reduced export/transport of cysteine to brain
Reduced neuronal GSH synthesis increased
sensitivity to heavy metals apoptosis Integrity
of the gut epithelium compromised
Increased mucosal membrane permeability
malabsorption of dipeptides Altered T-cell
subpopulations Increased CD4/CD8
Autoimmunity
57
Putting it all into Perspectivewe see what we
know
Cellular Metabolic Pathways
You Are Here
58
OVERALL CONCLUSION OCTOBER 2004
The abnormal metabolic profile in children with
autism is consistent with the abnormal genetic
profile and strengthens the hypothesis that a
genetic susceptibility to oxidative stress and
reduced methylation capacity may predispose these
children to the neurologic, immunologic, and
gastrointestinal dysfunction that occurs with
autism.
59
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