Beyond Methylation:

Thimerosal’s Impact on DNA and Sulfur-Dependent Redox in Autism

A New Toxic/Sulfur/Genetic Impact Theory And Pathway of Recovery

Presented byMichael Lang, M.F.A.

AutismOne Conference, Chicago©May, 2005

Introduction-Research History

Autism is likely caused by the “live” actions of thimerosal mercury (Hg) in tandem with prior “envirogenetic” susceptibility on familial DNA from cumulative, multi-generational effects of metal, agricultural and other chemical toxins.

The groundbreaking research of Drs. James, Neubrander, Deth, Boris, & Goldblatt cite mercury-initiated impairments in transsulfuration & methylation processes as primary impacts in Autism Spectrum Disorders.

Introduction-Research HistoryDrs. Amy Holmes and Boyd Haley, and Mark Blaxill, conducted a “first cut” baby hair study and found lower levels of mercury in autistic samples than controls. They postulated Autistic children may have a susceptibility to impaired mercury detoxification.

A scientifically thorough follow-up study conducted at M.I.T. fully confirmed these findings.

Dr. H. Vasken Aposhian theorized that Autism may be caused by a genetic compromise in “efflux” or transport phase of Hg detox, similar to Wilson’s Disease, which creates toxic accumulations of copper.

Hg, Sulfur, Redox, & DNA Damage Mercury damages brain neurons, pervasively disregulates enzyme, protein, lipid cell membrane and methylation functions. Mercury also most significantly impacts sulfur-dependent processes by binding to sulfur, severely disrupting its functions.

Mercury inhibits processes like methylation due to disruption of sulfur-dependent precursor proteins via a slowing of their electron transfer/redox processes, initiating the biochemical cascade of ASD symptoms.

Studies have established that Hg and other toxins cause major oxidative damage, lesions and single-strand breakage on human DNA. Hg further inhibits sulfur-dependent DNA repair of damage it causes.

This presentation is an extensive review of biochemical research papers that proposes a new unifying theory of how recycling or redox of biochemical processes governed by iron-sulfur protein clusters are a likely primary impact biochemistry in the effects of mercury toxicity.

This theory provides a deeper biochemical basis and connectivity the for oxidation, methylation, and Hg efflux defects cited by Drs. James, Neubrander, Deth, Boris, Goldblatt, & Aposhian.

A New Origin Impact &

Genetic Susceptibility Theory

Sulfur Chemistry As a Primary Hg Impact And Recovery Pathway Iron-sulfur proteins provide indispensable “Administrative Assistant” functions in transferring crucially needed electrons from one biochemical interaction to another.The “ Metabolic CEOs” can then complete processes they oversee to maintain the body’s collective “Gross National Product,” keeping the biochemical “economy” robust.

“Downsize” several seemingly benign biochemical “Admin. Assistants,” then routinely overwork those who remain to see how little work “CEOs” do on their own!

Without optimal reduction or recycling via unsung sulfur-based “Admin. Assistants,” essential biochemical work slows down, processes are diminished, and metabolic functions suffer from ongoing, pervasive impairments.

If we close in to a more elemental point of impact, we can develop a more effective understanding of how mercury damages the body via both live and ongoing damage to the brain, metabolic, & DNA functions & why recovered children may retain lifelong detox issues & oxidative stress.

For now, nutritional therapy can minimize many issues that may be eventually curable with more targeted DNA repair.

A way of testing for this active DNA damage is by urinary 8-hydroxydeoxyguanosine as a marker along with elevated oxidized NADP to reduced NADPH, oxidized cobalamin, and other redox process indicators would be important tests to confirm/deny this theory, possibly leading to more natural/targeted/effective Autism recovery protocols.

Hg Damage & Diminished Redox: Compromised DNA Code & Cure Target

RESEARCH HYPOTHESES:

1. Hg-initiated oxidative disruptions in iron-sulfur cluster-dependent redox biochemically precede impairments in glutathione synthesis via methylation pathway.

2. These sulfur disruptions are the primary reason levels of reduced or active glutathione (GSH) are low & elevated levels of oxidized or spent glutathione (GSSG) occur in Autism.

RESEARCH HYPOTHESES:

3. Hg-initiated oxidative DNA damage may facilitate ongoing oxidative stress, diminishing antioxidant & global redox/recycling processes.

4. Boosting antioxidants, using low dose/diverse sulfur & redox support, naturally & gently detoxing metals, & supporting DNA repair with iron-sulfur rich micro-algae may provide more comprehensive Autism recovery options.

What is Oxidation & Oxidative Stress?

Examples of familiar oxidation reactions:

Sliced apple browning: prevented by lemon juice

Nuts and chips: oxygen-generated rancidity/staleness

White wine: browns after oxygen enters bottle

Rust: oxidation of iron in combination with water

Antioxidants prevent or halt oxidative reactions & stress

What is Oxidation & Oxidative Stress? Think of oxidation of cells like the soiling of clothes or food left on dirty

dishes. Washing & drying clothes & dishes “reduces” them to a cleansed or “ready” state.

Oxidative stress occurs in the body when reserves of antioxidants are in insufficient form or supply to stem the cell and DNA-damaging effects of oxidizing chemistries.

Heavy oxidizers: Singlet oxygen & iron, agricultural toxins,

petrochemicals, heavy metals, & radiation.

Heavy casualties of heavy metal toxicity are oxidation of lipids, proteins and DNA.

Possible Mechanisms For Metal-Induced Oxidative Stress

Toxic MetalsPb, Cd, Hg, As

Damage to antioxidantdefense system

Depletion of Thiol status

Reactive Oxygen Species (ROS)

LIPIDS PROTEINS DNA

Lipid Peroxidation Protein Oxidation Oxidized Nucleic Acids

Cell Membrane Damage Protein Dysfunction Impaired DNA Repair

Cell Death MutagenicityCarcinogenesis

Adapted by Lang M, 2005 from Ercal et al, 2001

What is Redox & Why is it Important? Redox reactions always occur in pairs; as one substance is

oxidized, another is reduced.

The molecule that loses electrons in a biochemical reaction, is Oxidized; the molecule that gains those electrons is Reduced. These electron transfers are called Reduction/Oxidation or Redox reactions.

A way to remember this relationship: Redox Definition: Loss of Electron = Oxidation. Gain of Electrons = Reduction or remember: (LEO

the lion says GER)

What is Redox & Why is it Important?

Redox moves electrons of oxygen, nitrogen, hydrogen, and other elements from one process where they are reacted or will be disruptive if they remain and also delivers them to other processes where they can be used, converted, or eliminated from the body.

Redox processes work like interactive circular bucket brigades in biochemistry, transferring vital electrons from one cycle to another to maintain the thousands of biological processes occurring simultaneously within living organisms.

Iron-Sulfur (Fe-S) Protein Clusters

Some molecules that are oxidized & reduced during redox are called electron transfer proteins and are essential assistants in methylation & transsulfuration, & myriad metabolic processes.

The most prevalent electron transfer proteins are Nicotinamide Adenine Dinucleotide Phosphate Hydride (NADPH), Flavin Adenine Dinucleotide (FAD), & Flavin Mononucleotide (FMN).

FAD and FMN can redox and revitalize NADPH & are iron-sulfur protein cluster based enzymes that utilize cysteine & elemental sulfur as significant structural/reactive components.

4Fe-4S Iron-Sulfur Clusters

Fe

S

Fe

FeFe

S

S

S

Cys S Cys S

Cys S

Cys

S

Iron-Sulfur clusters function within electron transfer proteins like FAD, FMN, and ferredoxin. They are believed to have been a primary component of “Primordial Soup” and are a chemical link between the biological and non-living physical matter of our planet.

S

Fe

FeFe

S

S

S

Cys S Cys S

Cys S

4Fe-4S Iron-Sulfur Clusters with Reacted Iron-Cysteine Displaced

The reactive Fe-S-Cys atom reacts with atoms of binding elements & is displaced until replaced via redox by a compounded Fe-Cys molecule. Hg hobbles this redox & replacement, slowing rapid electron transfer, backing up oxidized metabolites, & disrupting metabolic processes like methylation.

Redox-Dependent Methylation

ALL the pivotal process steps in the synthesis of methionine into glutathione found to be impaired in Autism in James’ and Neubrander’s research are dependent upon NADPH, FAD, or FMN redox.

Mercury-inhibited electron transfer in the iron-sulfur cluster proteins FAD/FMN constitutes a far more comprehensive scenario for how mercury and other toxins affect methylation and other processes and induce chronic oxidative stress.

Hg-impaired redox would create pervasive disruptions but are only one of the many sulfur impairments mercury would cause in the body.

Fill

Interactive Circular Bucket Brigades

Dump

DumpFill

FillDump

Dump

Fill

Dump

Fill

Empty

Full

One dumps into another

Dump

FirstRecycling/

RedoxProcess

SecondRedox

Process

A Simplified Reduction & Oxidation or Redox Model

ThirdRedox

FourthRedox

FifthRedox

PrimaryChemicalProcess

HOMOCYSTEINE

FAD(H)(Fe-S)

Methionine Synthase

(MS)

MethionineSynthase

Reductase(MSR)

FMN/FADIron-sulfur

(Fe-S) protein coenzymes based on

Riboflavin/B2

Fe-S Dependent Transsulfuration of Homocysteine to Methionine

LEGEND

NADPH

Transsulfuration

Methionine Recycling

NA

DP

FMN(Fe-S)

Methyl-Cobalamin

NADP Nicotinamide Adenine Dinucleotide Phosphate

NADPH NADP reduced with Hydrogen molecule

FAD Flavin Adenine NucleotideFMN Flavin mono-Nucleotide

Hg

5-METHYL FAD(H)

(Fe-S)

5, 10MetheleneTetrahydro-

FolateReductase (MTHFR)

FMN/FADIron-sulfur

(Fe-S) protein coenzymes based on riboflavin

Reduction of 5,10-Methylene

Tetrahydrofolate by MTHFR

LEGEND

NADH

TETRAHYRDOFOLATE

Folate & DNA

Synthesis

NA

DKreb’sCycle

FMN(Fe-S)

NADH

NADP Nicotinamide Adenine Dinucleotide Phosphate

NADPH NADP reduced with Hydrogen molecule

FAD Flavin Adenine NucleotideFMN Flavin mono-Nucleotide

Hg

Primary & Secondary Pathway of MS Redox

The primary pathway of redoxing or recycling Methionine Synthase is via Methionine Synthase Reductase, with secondary redox accomplished by Methylcobalamin.

Both pathways are dependent upon NADPH and/or FMN/FAD iron-sulfur dependent redox.

Primary & Secondary Pathway of MS Redox

Oxidation and redox impairments and not methylation disruptions are more likely the basis for low levels of reduced glutathione, methylcobalamin, & folate.

A more complete approach to the problem would involve finding the origin of the oxidative stress/redox impairments and correcting this at its point of genesis if possible.

Disruption of Iron-Sulfur Clusters

Chemical toxins known to interfere with Iron-Sulfur proteins by disrupting their normal electron transfer: Rotenone: mild pesticide developed

from legumes but a potent inhibitor Amytal: a barbiturate that also inhibits

folate, Vit. D, and calcium metabolism Piericidin A: a penicillin-like antibiotic

and a potent inhibitor Heavy Metals: Lead, Mercury,

Cadmium are all potent inhibitors

Hg’s Disruption of Iron-Sulfur Clusters

In plants, even highly diluted, submicromolar concentrations of Hg can completely inhibit NADPH-dependent redox within 15 minutes.

Other studies have shown that mercury completely disrupts the “ping pong” to and fro functions of NADPH.

NADPH

NAD+

Krebs Cycle Cellular Energy Metabolism & NADPH/FADH Redox

NAD+

NADPH

FAD

FADH2

GTP(ATP)

NADPH

Acetyl-CoA

NADCO2

CoA

Citrate

NADPH

NAD+

IsoCitrate

Alpha-Ketoglutarate

Succinyl Co-A

Succinate

Fumarate

Malate

Oxaloacetate

Pyruvic Acid

Iron-Sulfur Cluster Proteins & The Kreb’s Cycle

NADPH & FADH are both reduced via and exert a

reactive influence within the Kreb’s Cycle of cellular energy production.

It is very likely that specific anomalies of Kreb’s Cycle acids in organic acid tests of Autistic children like pyruvate, citrate, alpha-ketoglutarate, succinate, or malate could be directly attributed to diminished electron transfers from Hg-impacted iron-sulfur proteins that cause biochemical “traffic jams.”

Iron-Sulfur Clusters and Metabolic Impacts:

The hidden role of iron-sulfur

protein-dependent redox in methylation transsulfuration pathway impairments cited by Drs. Jill James, James Neubrander, & Richard Deth.

0

1

2

3

4

5

6

7

8

ASD Before ASD After Controls

Total Glutathione

0

0.1

0.2

0.3

0.4

0.5

0.6

ASD Before ASD After Controls

Oxidized Glutathione

0

5

10

15

20

25

30

ASD Before ASD After Controls

Ratio of Reduced Glutathione to Oxidized Glutathione

Jill James’ Transsulfuration Research

51% of controls 72% of controls183% of controls 160% of controls

34% of controls 54% of controls

% of change post supplementsTotal glutathione: +21%Oxidized glutathione: -23%(Still 60% higher than controls!)Ratio of GSH to GSSG: +20%(Still 46% lower than controls!)Small change: Reduced GSH…Why? Low redox of oxidized GSSG to GSH!

(GSH) to (GSSG)

(GSSG)

SAM

SAH

Adenosine

Methylation of DNA, RNA, proteins, membrane phospholipids, creatine

Homocysteine

Vitamin B6

Cystathionine

Methionine

Cysteine

Reduced Glutathione

Protein Synthesis

MAT

MTASE

SAHH

CBS

Choline

Betaine

BHMT

MS

B12

THF

5-CH3THF

MSR

Vitamin B6

Inhibits MTase when elevated

NADPH-Dependent Transsulfuration of Methionine to Glutathione

KEY

BHMT- Betaine-homocysteine methyl transferaseCBS - Cystathione beta synthaseFAD - Flavin Adenine DinucleotideFMN - Flavin Mono NucleotideMAT - Methionine adenosyl transferaseMS - Methionine synthaseMSR - Methionine synthase reductaseMTASE - Various methyltransferasesNADPH - Nicotinamide adenine dinucleotide phosphateSAM - S-adenosylmethionineSAH - S-adenosyl homocysteineSAHH - S-adenosyl homocysteine hydrolaseTHF - Tetrahydroflate5-CH3THF - 5-methyltetrahydroflateFe-S - Iron-Sulfur cluster involvement

+- = low

levels

James’ & Neubrander’s Findings

= high levels

--

--

-

-

-

-

-

+

NADPH

NADPH

Fe-S

NADPH

FMN

FADH

NADPH

Oxidized Glutathione

++

Glutathione Reductase

NADPH

FADH

+

Adapted by Lang M, from James J, DAN! Conference, 2003

Redox-Dependent Methylation

ALL the pivotal process steps in the synthesis of methionine into glutathione impaired in Autism in James’, Deth’s, & Neubrander’s research are dependent upon NADPH/FADH/FMN redox.

Mercury-inhibited electron transfer in iron-sulfur cluster proteins constitutes a far more comprehensive scenario for how mercury and other toxins affect methylation and other processes and perpetuate chronic oxidative stress.

Hg-impaired redox would create pervasive disruptions but are only one of the sulfur impairments mercury would cause in the body.

GSSGGSH

NADP

NADPH

Protein-SSG Protein-SH

H2O2H2O

(Oxidized Glutathione)

ThiolTransferase

GSH Peroxidase

(Reduced/ Reactivated Glutathione)

GSSGReductase

NADH-Dependent GSSG Reductase Recycling of Oxidized Glutathione (GSSG) Back to its Reduced

Form (GSH)

Dr. Neubrander’s Protocol of Methylcobalamin Injections:

How do they help metabolically and can they balance disrupted methylation pathways, reduce oxidative stress and maintain a more normal balance of reduced glutathione (GSH) to oxidized glutathione (GSSG)?

Cob(I)alamin Cob(II)alamin Methyl-cobalamin

Oxidation/Reactivation

Catalytic Cycle

HomocysteineMethioni

ne

Methyltetrahydrofolate

Hydrofolate

NADPH

NADPH

Adapted by Lang, M. from Olteanu, H. et al. J. Biol. Chem. 2003;278:38310-38314

NADPH-Dependent Methylcobalamin Redox

& Fe-S Redox Protein+AdoMet

NADPH

Why Do Methylcobalamin B-12 Injections Help Autistic Children with Methylation

Impairments?

Chronic redox impairments would leave biochemical processes including, but not limited to, methylation shortchanged and functioning at a diminished rate. These backups would result in higher levels of oxidized forms of glutathione and cobalamin (B-12), to name just a few.

Why Do Methylcobalamin B-12 Injections Help Autistic Children with Methylation

Impairments?

Inhibition of methionine synthase from diminished redox from NADPH/FADH/FMN and methylcobalamin would impact the recycling of homocysteine into methionine, significantly inhibiting glutathione synthesis. Giving B-12 injections would provide an end of process “jumper cable” solution for the secondary support of methionine synthase that recycles homocysteine back into methionine.

Methylcobalamin & Redox

NADPH’s redox of cobalamin to methylcobalamin is yet another example of the role of iron-sulfur-dependent redox of methylation chemictry.

If NADPH itself is not redoxed effectively, by iron-sulfur dependent redox, then it cannot in turn convert oxidized or less active cobalamin into reduced or active methylcobalamin, thus creating another metabolic logjam.

Inhibited NADPH-dependent methylcobalamin redox = inhibited Methionine Synthase, which = incomplete recycling of homcysteine back into methionine which = less available glutathione.

Research Establishing Redox Control of Transsulfuration

Defects in Auxiliary Redox Proteins Lead to Functional Methionine Synthase Deficiency Journal of Biological Chemistry, Aug 1997

Redox control of the transsulfuration & glutathione biosynthesis pathways. Current Opinion in Clinical Nutrition & Metabolic Care, Jan 2002

The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes. Biochemistry, Oct 2000

Redox regulation of homocysteine-dependent glutathione synthesis. Redox Report, Aug 2003

Research Establishing Redox Control of Transsulfuration

Molecular dissection of human methionine synthase reductase: determination of the flavin redox potentials in full-length enzyme and isolated flavin-binding domains. Biochemistry, Apr 2003

Molecular basis for methionine synthase reductase deficiency in patients belonging to the cblE complementation group of disorders in folate/cobalamin metabolism Human Molecular Genetics, Oct 1999

5-,10-methylene tetrahydrofolate

5-methyl-tetrahydrofolate

NADPH/FAD-Dependency: Deth’s Methylation Research

5-,10-methenyl tetrahydrofolate

5-formino-tetrahydrofolate

5-formyl tetrahydrofolate

APP + Pi

ATP

tetrahydrofolate

10-formyl-tetrahydrofolat

e

formate

ATP

ADP + Pi

Serine hydroxymethyl transferase

Serine Glycine H20

KEY

ATP - Adenosine triphosphateADP - Adenosine diphosphateFADH - Flavin Adenine Dinucleotide HydrideFMN - Flavin MononucleotideNADH - Nicotinamide adenine dinucleotide hydride NADPH - NADH (reduced)NADP - Nicotinamide adenine dinucleotide phosphate

5, 10-methylene tetrahydrofolateReductase (MTHFR)

NAD+

NADH

FADH

5-, 10-methenyl tetrahydrofolatedehydrogenase

NADP+

NADPH

10-methenyl Tetrahydrofola

te cyclohydrase

NADP

10-formyl-tetrahydrofolat

e dehydrogenase

NADP

P5P/B6

5, 10-Methylene tetrahydrofolate

7, 8-Dihydrofolate

Tetrahydrofolate

Deoxyuridine 5-monophosphate

Deoxythymidine 5-monophophate

Dihydrofolate Reduction, Thymidine & DNA Synthesis, & NADPH

DNA Synthesis

Serine

Glycine Serine Hydroxymethyl Transferase

10-Methylene tetrahydrofolate reductase

(MTHFR)

NADPH FADH FMN

Dihydrofolate reductase

NADPH FAD

NADPNADPH(Glycine CleavageSystem)

Research Establishing Redox Involvement in MTHFR

Methylenetetrahydrofolate reductase. Steady state and rapid reaction studies on the NADPH-methylenetetrahydrofolate, NADPH-menadione, and methyltetrahydrofolate-menadione oxidoreductase activities of the enzyme. Journal of Biological Chemistry, Sep 1983

Purification & characterization of methylenetetrahydrofolate reductase from human cadaver liver. Journal of Biological Chemistry, Sep 1984

Purification and properties of NADH-dependent 5,10- methylenetetrahydrofofolate reductase (MetF) from E.coli. Journal of Bacteriology, Feb 1999

Purification and properties of 5,10-methylenetetrahydrofofolate reductase, an iron-sulfur flavoprotein from Clostridium Formicoaceticum. Journal of Biological Chemistry, Sep 1984

Hg-Initiated Oxidative DNA Damage: Do Chronic Redox Impairments in Autism Have a Genesis In Damaged Genetic Code?

Genomic research has shown that DNA mutations at the binding sites of Nicotinamide Adenine Dinucleotide Hydride, (NADPH), Flavin Adenine Dinucleotide (FAD, & Flavin Mononucleotide (FMN) are involved with Methionine Synthase Deficiency conditions via diminished redox of Methionine Synthase Reductase.

Iron-sulfur,DNA Mutations, and Methylation Chemistry

Cellular E. coli & Mammalian Methionine Synthase Redox Via

FMN/FAD/NADPH

FMN FAD NADPH

FMN Linker Region NADPHFAD

Methionine Synthase Reductase Binding Sites

FlavodoxinNADPH-Ferrodoxin

(Flavodoxin) Reductase

FMN Linker Region FAD NADPH

16del6

V54del

V56M

C405R

G487R

1554del7

L576del

1675del4

G554R

A129T

R114X

Intronic T to C

L333V

E. ColiMSR

KEY

FAD - Flavin adenine dinucleotideFMN - Flavin mononucleotideNADPH - Nicotinamide adenine dinucleotide phosphate (reduced)

HumanMSR

DNA mutations in Iron-Sulfur Cluster Dependent Enzyme Binding Sites in Methionine Synthase Reductase (MSR)

Schematic of MSR showing 13 reported human DNA mutations in relation to MSR binding sites on chromosome 5p15.2-p15.3

Reported MSR enzyme mutations involved with MS(R) deficiency

Adapted by Lang, M from Wilson, A et al Human Molecular Genetics, 1999

Research on Hg’s Effects In Oxidative DNA Damage

Mercuric chloride damages cellular DNA by a non-apoptotic mechanism.

Mutation Research, Oct. 2000

Increased oxidative DNA damage, as assessed by urinary 8-hydroxy-2'-deoxyguanosine concentrations, & serum redox status in persons exposed to mercury. Clinical Chemistry Apr. 2005

Interactions of Hg(II) ions with DNA as revealed by CD measurements.Nucleic Acids Research Mar 1977

Research on Hg’s Effects In Oxidative DNA Damage

Oxidative damage to nucleic acids in motor neurons containing

mercury. Clinical Chemistry, Apr. 2005

Time course assessment of methylmercury effects on C6 glioma cells: submicromolar concentr. induce oxidative DNA damage and apoptosis.Journal of Neuroscience Research, Dec 2002

Research:Hg’s DNA Damage

& Inhibition of DNA Repair

Analysis of metal-induced DNA lesions and DNA-repair

replication in mammalian cells. Mutation Research, Mar-Apr 1984

Correlations of DNA strand breaks and their repair with cell survival following acute exposure to mercury and X-rays. Molecular Pharmacology, Jul 1983

Research on Hg’s Inhibition of DNA Repair

Use of mammalian DNA repair- deficient mutants to assess the effects of toxic metal compounds

on DNA. Biochemical Pharmacology May 1984

Differences in the effects of Hg (II) on DNA repair induced in Chinese Hamster ovary cells by ultraviolet or X-rays. Molecular Pharmacology, Feb, 1986.

How to Prevent or Repair

Oxidation-Damaged DNA

Glutathione & ascorbate are negatively correlated with

oxidative DNA damage in human lymphocytes Carcinogenesis, Apr, 1999

Oxidative DNA damage in human lymphocytes: correlations with plasma levels of tocopherol & carotenoidsCarcinogenesis, Feb 2000

Lymphocyte Oxidative DNA Damage and Plasma Antioxidants in Alzheimer DiseaseArch Neurol, May 1, 2002;

Fe-S Cluster’s Repair of Oxidation-Damaged DNA

A Role for iron-sulfur clusters in DNA repair. Curr Opin Chem Biol. Apr 2005

Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding. Proceedings, National Academy of Sciences. Mar 1995

Atomic structure of the DNA repair [4Fe-4S] enzyme endonuclease III. Science. Oct 1992

A substrate recognition role for the [4Fe-4S]2+ cluster of the DNA repair glycosylase MutY. Biochemistry. May 1998

Nutritional Protocols Used Successfully For Autism To Assist in Redox & Hg Detox:

What biochemical processes do they support and why do they work?

Reductase (recycling) of

GlutathioneACTIVE GSH

Creates GSH peroxidase: protects cell membranes from lipid peroxidation, toxic metals, vital to cell-mediated immunity

Bre

aks

do

wn

into

ta

uri

ne

& c

yste

ine

Goes on as

l-cysteine &

breaks down

into taurine

TAURINE

ME

TH

ION

INE

CYSTEINE(NAC)

SULFUR

BEARING

AMINO A

CIDS

Direct

phase II

detox o

f exce

ss

fatty

acid

s. pro

mote

s flow o

f bile

GLUTATHIONE

GL

YC

INE

CYSTEINE

GL

UT

AM

IC

AC

ID

Alp

ha K

eto

Glu

tatate (AK

G)

DetoxesHg, Pb, Au, Cd

Best way to recycle/ raise plasma GSH

AO, Protects cells, supports immune system

Metal-detox, enzyme &

immune support

Strong AO, clears free

radicals

Selen

ium

A N

T I - O

X I D

A N

T S

Vit. E

ZIN

CV

it. CA

LA

Sulfur-bearing AO, enhances

function of GSH, vit A, C, E, Zn, Se

Hg Detox, only AO

both fat & H20 soluble

Niacin

(B-3)

Riboflavin (B-2)Used for GSH reductaseenzymerecycling R

ecycles

GS

HMethionine & molybdenum

for phase II sulfation detox

(phenols)

Sulfoxidation of

Cysteine

Helps break down

cysteine into

taurine

Crucial for methionine breakdown to cysteine/methylation

pathway

Crucial vitamin

in enzyme nthesis

VITAMIN B-6 (Pyridoxine) Magnesium

B-6 in excess is neurotoxic & inhibits sulfation

B-6 B-6 & Mg activate wide

range of detox enzymes

Create active sulfotransferases

(PST) & helps construction of

PAP’s

Initiation ofmercapturic

GSH pathwayof urinary toxin

clearance

Crucial mineral in enzyme &

GSH synthesis

Used for

Glutathione synthesis

Thiamin(B-1)

ME

TH

YL

DO

NO

RS

Ch

oli

ne

& I

no

sit

ol

Fo

lic

ac

id

& v

it.

B-1

2D

MG

&T

MG

GL

YC

INE

Folinic & B12 recycle

homocysteine to methionine

Choline assists TMG in recycling

homocysteine to Methionine

Glycine to GSH

synthesis Betaine

balances HCy to methionine

All Roads In Effective Nutritional ASD Protocols

Lead to GlutathioneFrom the first use of B-6 & Magnesium to Dimethylglycine, Vitamin C, Zinc, and other Antioxidants, N-Acetyl Cysteine, Alpha Lipoic Acid, TTFD/Allithiamine, to today’s use of B-12 and Folate, the primary metabolic benefit has been the enhanced synthesis and redox/recycling of Glutathione.

Supplement Protocol for Enhancing Glutathione Synthesis and Recycling

(Redox): B-VITAMINS

Avoid tablets and capsules if possible due to poor breakdown and assimilation with sub-optimal gut structure/function. Liquids are better absorbed than other forms and provide 2-4x better assimilation. Use optimal daily dosages of all the B-vitamins in the following median levels for a 50 lb. Child, given 3x/day in divided dosages:

Vit B-1 (Thiamin Hydrochloride - 20-35 mg (GSH synthesis) Vit B-2 (Riboflavin-5-Phosphate) - 20-35 mg (Fe-S/flavoprotein building block) Vit B-3 (Niacinamide) - 15-25 mg (GSH redox)Vit B-3 (Inositol Hexaniacinate) - 20-30 mg (GSH redox)Vit B-5 (Calcium Pantothenate) - 100-150 mg (Acetyl Coenzyme A support) or - sulfated B-5:Pantetheine - 50-75 mg (Enhanced Acetyl Coenzyme A support)Vit B-6 (Pyridoxyl-5-Phosphate) - 15-35 mg (Cysteine, GSH, detox enzyme synthesis) or - Pyridoxine Alpha Ketoglutarate - 3-75 mg

(if P-5-P intolerance)Vit B-12 (Methylcobalamin) sublingual form - .5-2 mg (Methylation “jumper cable”)Folinic Acid (5-methylTHF), sublingual form - .5-2 mg (Methylation “jumper cable”)Biotin - 300-500 mcg (Sulfur-bearing catalyst)Choline (Citrate) - 60-100 mg (Lipid cell membrane/myelin & detox support)Inositol (FCC) - 80-100 mg (Lipid cell membrane support)

Supplement Protocol for Enhancing Glutathione Synthesis and Recycling

(Redox): ANTIOXIDANTS

Use optimal daily dosages of all the antioxidants in the following levels for a 50 lb. child, administered 3x/day in divided dosages:

•Avoid synthetic vitamin A acetate/palmitate and synthetic vitamin E dL-tocopherols!!!Both are either synthesized from petroleum or contain petroleum-derived preservative agents and can also block the assimilation of natural forms of vits A & E-ASK BEFORE YOU BUY!!

Pro-vitamin A Mixed carotenoids - 6,000 IUor QUALITY fish oil* (Redox/gut/immune support)

Vit C as Calcium Ascorbate - 750 mg-1 gr.3x/day (GSH/Redox support)Lemon Bioflavinoids - 175-300 mg (Anti-inflammatory & increases Vit. C potential)Vit D-3 (Cholecalciferol - 200-300 IU (Enhances calcium metabolism & uptake)Vit E (Natural tocopherol)*- 125-200 IU (GSH redox & cell membrane antioxidant

TMG - 100-750 mg (GSH precursor/methyl donor)L-Glycine - 100-250 mg (GSH precursor)N-Acetyl Cysteine - 10-30 mg (GSH precursor & MT synthesis)

Supplement Protocol for Enhancing Glutathione Synthesis and Recycling

(Redox): MINERALS

Use optimal daily dosages of all the minerals in the following levels for a 50 lb. child, administered 3x/day in divided dosages:

Calcium (Kreb’s Cycle Chelates) - 1200 mg (Prevents cellular uptake of Hg) Chromium (Kreb’s Chelates) - 100 mcg (Blood sugar balance) Magnesium (Kreb’s Chelates)- 375 mg (GSH & MT synthesis & redox) Manganese (Succinate) - 2-3 mg (GSH & MT synthesis & redox) Molybdenum (Kreb’s Chelates) - 200 mcg (Sulfur process catalyst) Potassium (Alpha-Ketoglutarate) - 100 mg (Nerve cell function & ammonia detoxSelenium (Selenomethionine) - 100 mcg (Hg detox & GSH peroxidase cell defense)Zinc (Kreb’s Chelates) - 50 mg (Hg detox, MT synthesis, & GSH redox)

Supplement Protocol for Enhancing Glutathione Synthesis/Redox, & Heavy

Metal Detoxification ACCESSORY NUTRIENTS

Use optimal daily dosages of all the minerals in the following levels for a 50 lb. child, administered 3x/day in divided dosages:

GAG synthesis/intestinal detox: Calcium D-Glucarate - 100 mg

Gut inflammation support:Methylsulfonylmethane (MSM)* - 200 mg

DMAE for attention/focus - 20-35 mg

NADH: One 10 mg sublingual taken 20 min. before breakfast(for support of NADPH-dependent processes)

Glutathione as sublingual/liposomal- 300-500 mg, 20-60 min.before meals

Nanocolloidal Detox Factors (NDF) A natural, chorella-based chelation option

* Use only U.S.-made MSM - some foreign material is “questionable”

Supplement Protocol for Enhancing Glutathione Synthesis and Recycling

(Redox): HERBS

Ashwaganda Root (Withania somnifera) Supports detoxification via its GSH redox support.

Burdock Root (Arcticum lappa) Promotes liver cleansing, bile flow as well as detoxification via its GSH redox support

Chinese Astragalus Root (Astragalus membranaceus) Supports detox. via GSH redox support.

Fennel Seed (Agastache foeniculum) Sulfur source.

Ginkgo Biloba Leaf (Gingko Biloba) Increases cellular GSH and GSH S-transferase

Gotu Kola Leaf (Centella asiatica) Raises levels of GSH.

Milk Thistle Seed (Silibum marinum) Sulfur source. Assists GSH redox support.

Chinese Sarsaparilla (Smilax Glabra) Used in Chinese medicine to detoxify mercury. Assists in GSH redox and cellular thiol status.

Schisandra Fruit (Schisandra chinensis) Liver protectant. Supports detoxification via recycling of glutathione.

Summary1. Multi-generational exposure to toxins

that has affected familial DNA may have set the stage for a susceptibility factor via iron-sulfur-cluster disruptions, increasing the oxidative damage caused by Thimerosal mercury.

2. Mercury is the “rock thrown,” but sulfur is the “window” that has broken. This scenario is very likely to be the core issue of the pervasive disregulation of metabolism we are seeing in Autism.

Summary3. Hg-initiated oxidative disruptions in iron-sulfur cluster-dependent redox biochemically precede impairments in glutathione synthesis via the methylation pathway.

4. These sulfur disruptions are the primary reason levels of reduced or active glutathione (GSH) are low & levels of (GSSG) oxidized or spent glutathione are elevated in Autism.

Summary5. Hg-initiated oxidative DNA damage may facilitate ongoing oxidative stress, crippling antioxidant & global redox processes. This is our biggest challenge in recovering and maintaining the health of our children.

6. Boosting antioxidants, using low dose/varied sulfur & redox support, naturally & gently detoxing metals & supporting DNA repair with iron-sulfur rich Chorella/Spirulina may provide more comprehensive Autism recovery options.

For Our Children…