OXIDATIVE STRESS

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OXIDATIVE STRESS AUTISMA Roadmap for Effective
Treatment

• William J. Walsh, Ph.D.
• Pfeiffer Treatment Center
• Warrenville, IL

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Pfeiffer Treatment Center

• Outpatient medical facility
• 20,000 patients from all 50 states and 75 foreign
  countries.
• Collaboration between medical doctors and
  scientists.
• Individualized Biochemical Therapy
• Scientific Research
• 501c3 Public Charity

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Pfeiffer Chemistry Database

• 10,000 Behavior ADHD
• 4,600 Autism
• 3,500 Schizophrenia Bipolar
• 3,200 Depression

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Pfeiffer Autism Database

• About 90 to 150 assays of chemical factors in
  blood, urine, or hair for more than 4,600
  patients
• More than 500,000 separate chemical analyses

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Database Analysis

• Major biochemical differences exist between
  autistics and the general population,
• Autism chemical imbalances are generally more
  severe than those for violent behavior, ADHD,
  depression, bipolar disorder, and schizophrenia,
• Female autistics have more disordered chemistry
  than male autistics.

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High Incidence Biochemical Abnormalities in Autism

• Elevated serum copper
• Elevated toxic metals
• Depressed zinc
• Malabsorption/Maldigestion
• Undermethylation
• Pyrrole Disorder

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Year 1999 Discovery

• Undermethylation present in more than 90 of
  autism-spectrum children.

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Year 2000 Discovery

• Greater than 99 of ASD patients exhibit a
  serious metal-metabolism disorder.
• Elevated Cu and depressed Zn are found throughout
  the autism spectrum.
• in most individuals, Cu Zn are homeostatically
  controlled by metallothionein proteins.
• Conclusion Depressed metallothionein activity is
  a
• distinctive feature of
  autism.

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Experimental Results and Statistical Analysis

• Mean Cu/Zn Ratio
• Autism Spectrum (N503) 1.63
• Controls (N25)
  1.15
• t 8.77 (two-tailed t test) p lt 0.0001
• American Psychiatric Association Annual Meeting
• New Orleans, 2001.

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Insufficient Ceruloplasmin Levels in
Autistic-Spectrum Patients

• 
  Autistics Controls
• Unbound Serum Cu 41 21
• Not bound to ceruloplasmin
• P lt 0.01
• Autistics exhibit excessive levels of loosely
  bound or free-radical copper.

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Abnormally Elevated Copper

• Depletes metallothionein glutathione
• Associated with inflammation excessive
  oxidative stress
• Can cause abnormal neurotransmitter levels.

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Impact of Copper on Dopamine and Norepinephrine
Neurotransmitters
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Animal Studies Impact of Cu Level on Dopamine
and Norepinephrine

• Rodents fed Cu-deficient diet reducing blood
  levels to 25 of normal,
• Brain tissue assayed for dopamine and
  norepinephrine.
• RESULT Norepinephrine/Dopamine Ratio
• Reduced by a Factor of Four.

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Why is Metallothionein Important?

• Required for development of brain cells and
  synaptic connections,
• Primary filter for Hg, Pb, and other metal
  toxics at intestinal and blood/brain barriers,
• Required for homeostasis of Cu and Zn,
• Supports immune function,
• Major antioxidant system in body brain.

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Oxidative Stress and Metallothionein

• MT has powerful antioxidant properties,
• MT is produced by the body in response to many
  forms of oxidative stress,
• MT functions in tandem with GSH and Se in
  sequestering Hg, Pb, and other toxic metals.
• -- MT is a magnet for mercury, but MT activity
  is weak in autism-spectrum children.

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The Role of Metallothionein in the Development of
Brain Cells

• MT-3 assists in the pruning of brain cells, which
  makes space for growth of new cells,
• MT-1 and MT-2 participate in the natural growth
  (development) of brain cells,
• MT-3 is the primary agent for termination of
  growth of fully-developed brain cells.

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Autopsy Studies Show Structural Abnormalities in
Autistic Brains

• Short, dense, undeveloped brain cells,
• Abnormalities observed primarily where MT levels
  are highest (amygdala, hippocampus, Purkinje
  cells, inferior olives, and pineal gland).
• Conclusion Autistic brains may have failed to
  complete the maturation process due to low MT
  levels.

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Natural Protection Against Mercury and Other
Toxic Metals

• 95 of ingested Hg, Pb, Cd is stopped by MT GSH
  at the intestinal mucosa.
• 80 of toxic metals entering portal blood stream
  become bound to MT GSH in liver.
• 90 of remaining toxic metals are sequestered at
  B/B barrier by MT GSH.
• MT GSH are also present in the brain and
  provide additional protection.
• MT is a magnet for Hg, Pb, Cd, etc., and
  once bound to MT, toxic metals are inactive.

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Teamwork Between Metallothionein, Glutathione,
and Selenium

• GSH is first line of defense against Hg, Pb, etc,
  but has limited capacity for toxic metals.
• When gt 10 of GSH is bound to toxic metals,
  additional toxics are transferred from GSH to MT.
• Se increases kinetics of the GSH/MT antioxidant
  system by more than 50.
• In the case of major exposures, most of the toxic
  metals depart the body bound to MT.

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Distinctive Features of Autism

• Strong genetic predisposition
• Onset after environmental insult
• High oxidative stress
• Undermethylation
• Incomplete brain maturation

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Genetic Aspects of Autism

• Strong genetic predisposition
• -- Higher concordance in siblings
• -- 60 to 80 concordance in identical twins
• Influence of environmental factors
• -- Identical twin concordance not 100
• -- Major differences in many identical twins.

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QUESTION How Can There Be An Epidemic of a
Genetic Condition?

• ANSWER
• The genetic defect involves a weakened ability
  to cope with environmental stresses

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Timing of Environmental Insults is Important

• In Utero
• Autism evident at birth. Greater severity of
  symptoms. Mental retardation often present.
• After Birth
• Regressive autism. Symptoms depend on
  developmental stage during insult.

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Severity of Environmental Insult Is Important

• Example Disruption of key brain proteins
• during development of speech
  center.
• Mild insult results in speech delay.
• Severe insult results in mutism.

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Poly-Gene Nature of Autism

• Current consensus that autism results from many
  genetic defects, rather than from a single gene.
• A common factor in these genetic defects may be
  diminished ability to cope with oxidative stress.
  

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What is Autism?Oxidative Stress Theory of Autism

• Genetic tendency for depressed GSH, MT, Se, etc
  at intestinal and blood/brain barriers,
• Inability to prevent Hg, Pb, Cd, and reactive
  oxygen specie from invading the brain.
• -- destruction of brain cells
• -- interruption of brain maturation
  process

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Consequences of Oxidative Stress Mirror Classic
Symptoms of Autism

• Hypersensitivity to Hg and other toxic metals
• Hypersensitivity to certain proteins (casein,
  gluten, etc)
• Poor immune function
• Disruption of the methylation cycle
• Inflammation of the brain G.I. tract.
• Depletion of glutathione metallothionein
• Excessive amounts of unbound copper

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Consequences of Oxidative Stress in the G.I. Tract

• Destroys digestive enzymes needed to break down
  casein gluten proteins,
• Promotes candida/yeast levels,
• Diminishes Zn levels and production of stomach
  acid,
• Produces inflammation,
• Ineffective barrier to toxic metals at the
  intestinal mucosa.

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Most Popular Autism Therapies Enhance
Anti-Oxidant Protection

• Chelation with DMSA, DMPS, EDTA, etc.
• Methyl B-12
• Metallothionein Promotion
• Transdermal or Injected Glutathione
• Zn, Se, CoQ-10, Taurine, Vitamins A,C,D,E
• Alpha Lipoic Acid
• Risperdal

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Undermethylation Can Result in Excessive
Oxidative Stress

• An efficient SAM methylation cycle is needed for
  production of GSH, MT, and cysteine, which are
  primary antioxidants in the body.
• Undermethylation can result in incomplete
  sequestering of toxic metals in the liver which
  can deplete the body of GSH, MT, and Zn.

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Oxidative Stress Can Result In Undermethylation

• Oxidative stress results in depletion of GSH,
• GSH is necessary for efficient removal of
  adenosine in the SAM Methylation Cycle,
• GSH promotes the reconversion of homocysteine to
  methionine.
• Oxidative stress can be (1) the cause of
  undermethylation, or (2) the result of
  undermethylation.

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Mercury Questions

• What of autism cases are triggered by Hg?
• Can old Hg stay in the brain and cause
  continuing damage?
• How serious is the continuing daily exposure to
  Hg from the environment?

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Chelation and Oxidative Stress

• DMSA and DMPS are powerful antioxidants.
• Chelation can provide antioxidant benefits even
  if toxic metals are not present.
• For many patients, the primary benefits of
  chelation result from antioxidant properties, and
  not from removal of Hg or other metals.
• Antioxidant benefits from chelation appear to
  fade away after about 2-4 weeks.

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Primary Benefits of Chelation

• Rapid removal of toxic metals from peripheral
  soft tissues blood,
• Powerful antioxidant

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Limitations of Chelation

• Does not fix intestinal or blood/brain barriers,
  rendering the patient vulnerable to future toxic
  exposures,
• Antioxidant benefits are temporary, lasting only
  2-4 weeks,
• May not remove toxic metals from the brain,
• Complicates Zn management.

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Pfeiffer Treatment Protocol

• Identification individualized treatment of
  biochemical imbalances,
• MT-Promotion therapy,
• Selective use of adjunct therapies
• - CF/GF diet
• - Normalization of intestinal flora
• - Clathration/Chelation protocols
• - Digestive enzymes
• - etc.

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MT Promotion Therapy

• Primary Objective
• Advances in cognition, socialization, and
  speech by enhanced development of immature brain
  cells and new synaptic connections.

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MT Promotion Therapy

• Secondary Objectives
• Elimination of toxic metals excess Cu
• Improved immune function
• Healing of the G.I. tract
• Reduced food sensitivities
• Improved behavior control

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MT-Promotion Formulation

• Generous amounts of Zn and GSH which are
  essential to induction and functioning of MT,
• Selenium, Vitamins B-6, C, E, which are known
  to promote MT,
• Supplements of the 14 amino-acid constituents of
  MT in the proportion they exist in MT proteins.

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Unique Advantages of MT-Promotion

• Directly aimed at development of brain cells
  new synaptic connections,
• Potential for permanently correcting the
  intestinal and blood/brain barriers,
• Restores the natural (and powerful) body system
  for coping with toxic metals,
• Potential for eliminating food sensitivities,
  yeast problems intestinal inflammation.

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MT Promotion Challenges

• Pre-loading with zinc is necessary to prevent
  temporary side effects,
• Building up tolerance to the MT Promoter
  formulation can be a slow process for some
  children,
• Commercial lab testing to determine MT status is
  in its infancy.

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MT Promotion Aids Zn Normalization
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A Strategy for Enhanced Cognition, Speech, and
Socialization

• Elimination of toxic metals and excessive
  oxidative stress,
• Behavioral therapy to stimulate development of
  brain cells and synaptic connections,
• MT-Promotion therapy to enhance maturation of the
  brain.

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Autism Research At Pfeiffer

• RBC Metallothionein Measurements
• Oxidative Damage Study (U. of Penn.)
• Oxidative Stress Study (U.C. Berkeley)
• Oxidative Damage Study (NIH Case Western
  Reserve)
• Hormone Study (BHARE Foundation)
• Treatment-Naïve Database Studies
• Assays of Hg, Pb, Cu, Zn, S, etc in
  Autism/Control Brain Tissues using APS
  Synchrotron X-Rays.

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Metallothionein Measurements

• RBC or WBC preferred since serum/plasma MT
  escalates dramatically during stress.
• Best results using chilled samples tubes,
  refrigerated centrifuge, dry-ice shipment.
• Proteolysis inhibitors recommended.

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Low Metallothionein Levels in Autismp lt 0.0092
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Oxidative Damage Study

• Collaboration between U. of Pennsylvania
  (Dominico Pratico) and Pfeiffer Treatment Center,
• Controlled double-blind study of oxidative damage
  in fats and vascular tissue,
• Treatment-naïve autistics and age/gender matched
  controls.
• Findings Higher oxidative damage to fats and
  vascular
• tissues in autistic subjects.
  Paper accepted by
• Annals of Neurology.

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Oxidative Stress Study U.C. Berkeley

• Treatment-Naïve Autistics and Age/Gender Matched
  Controls from Pfeiffer Treatment Center,
• Measurement of Multiple Oxidative Stress
  Oxidative Damage Factors,
• Genetic Studies Underway
• Early Findings Strong evidence of increased
  oxidative
• stress/damage in
  autistic subjects.

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Oxidative Damage Study (CWRU)

• Treatment-naïve autistics/controls from Pfeiffer
  Treatment Center,
• Measurement of levuglandins and pyrrole adducts
  at Case Western Reserve U,
• NIH funding
• Experimental results not yet available.

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Autism Hormone Study

• Objective
• To investigate possible differences in hormone
  factors between pre-puberty male autistics and
  controls. Significant results could lead to a
  hormone-balancing therapy.
• Funding provided by the BHARE Foundation

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Test Subjects Hormone Study

• Treatment-naïve male autistics,
• Age range, 3 to 8 years
• Age/gender matched controls.

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Hormone Study Results

• Striking depression of DHEA-s levels in autistic
  subjects compared to controls.
• Significantly higher SHBG levels in autistic
  subjects,
• No detectable differences in estrogen, estradiol,
  testosterone levels until age 5.

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Treatment-Naïve Database Studies

• Blood/urine/hair chemistries for ASD children
  with no history of antioxidant or biochemical
  treatments,
• Comparison with age/gender matched controls,
• Improved characterization of chemical
  abnormalities associated with autism.

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Sampling of Treatment-Naïve Results

• Extreme elevations of copper, urine pyrroles,
  toxic metals, oxidative damage parameters, and
  absolute basophils observed in autistics,
• Severe zinc deficiency,
• Female autistics exhibit more disordered
  chemistry than male autistics,
• Urine pyrroles correlate strongly with oxidative
  damage and zinc deficiency.

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Comparison of Elemental Levels in Autism
Control Brains

• Double blind, controlled study,
• 176 brain tissues 22 peripheral samples from U.
  of Marylands Autism Brain Bank,
• Elemental analysis for 16 elements, including Hg,
  Pb, Cu, Zn, and Se using high-brilliance photons
  at ANLs Advanced Photon Source),
• First elemental assays ever attempted for autism
  brain tissues.

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Autism/Control Tissue Array
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Brain Regions Studied

• Cerebellum
• Superior Cortex
• Deep Cortex
• White Matter
• Note 22 autistic 22 control samples from each
  brain
• region

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Duplicate Chemical Analyses Were Performed for 10
Peripheral Tissues

• Kidney Liver
• Skin Tonsil
• Bladder Thymus
• Prostate Gall Bladder
• Lung Salivary Gland
• Note Autistic Subjects Only

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Status of Brain Tissue Study

• Testing of 198 coded samples completed,
• Fairly uniform levels of chlorine, sodium,
  potassium observed for all tissues,
• Major variations in Hg, Pb, Ca, Zn and Fe for
  different test subjects,
• Data analysis underway.

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Summary

• Oxidative stress may be the decisive factor in
  autism-spectrum disorders.
• Treatment protocols aimed at development of brain
  cells and synapses appear highly promising.

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THANK YOU!

• William J. Walsh, Ph.D.
• Pfeiffer Treatment Center
• Warrenville, Illinois
• www.hriptc.org