Re:EWG Report ---- Autism report news release 12.13.04

[Guest guest]

http://www.ewg.org/reports/autism/execsumm.php

[Guest guest]

The implications of these findings extend well beyond thimerosal and

autism. Reduced antioxidant defense may characterize a group of

individuals who are demonstrably more sensitive to the effects of a

range of toxic chemical exposures, and shed light on increasing rates

of related learning and behavioral disorders.

My son was a participant in the follow up studies, not autistic, yet

had apraxia, major sensory issues, coordination problems, the

nutritional interventions he was given/supplements, reversed all of

his issues, this is a fact.So this goes way beyond just autism, a

friends child with CAPD,ADD was also a participant and he too is now

without any diagnosis of CAPD/ADD.

Colleen

>

> http://www.ewg.org/reports/autism/execsumm.php

>

>

>

[Guest guest]

Hello Everyone and Hello Colleen,

This EWG report is astonishing and I am surprised that the list

didn't go crazy over this very important finding which could affect

all of us on this list. My impression is that we need a serious

discussion of the report and its possible bearing on apraxia.

The report -- like all the ground-breaking research -- centers on

autism, but it states several times that it relates to other

neurodevelopmental disorders. It sounds like it could provide THE KEY

for the vast majority of our apraxic children. It explains so much --

the reason why so many kids with all kinds of neurodevelopmental

learning disorders live in the same neighborhoods (for example, why

three of the four children I know who were adopted from the same

orphanage in Bethlehem all have neurodevelopmental learning

disorders), why there has been such an explosion of

neurodevelopmental learning disorders over the past decades, why so

many neurodevelopmental learning disorders overlap and are co-morbid,

why non-autistic children have been helped and sometimes cured

through biomedical protocols designed for autistic children.

I need no convincing -- I believe that in a great number of cases,

the various disorders (the autism spectrum disorders, ADD/ADHD,

apraxia, sensory issues, some forms of cerebral palsy, some forms of

brain damage, some forms of microcephaly, etc.) are related and that

the glutathione/heavy metal connection is central. I don't need to

wait for the double-blind clinical trials to go out now and try this

new glutathione and methylcobalamin S. Jill protocol.

When Colleen says that her son was cured of his apraxia, sensory

issues and coordination problems, that another child was cured of

CAPD and ADD, we should all be clamoring to hear where we can sign

up. This is INCREDIBLE, and if you read the report, all the " Aha!

Yes! This is IT! " bells will go off in your head. At least for a

great number of children suffering from these neurodevelopmental

disorders. No, not all, but just very possibly a majority.

Colleen, is there any way that we can get from you the protocol that

Dr. followed? I want to know just what she did in the follow-

up study your son paricipated in. There is a little bit of

information in the last appendix of the 2004 second edition of

Children with Starving Brains by McCandless, but not

really enough to show to a doctor and say " Do this, please. " I'm

particularly interested in getting the protocol because I live

in the Netherlands now and have to basically " teach " a willing doctor

here what to do.

How many parents on this list have followed a DAN! protocol for their

non-autistic child? How many have had their child tested for heavy

metal poisoning -- with a hair strand test AND a pre- and post-

challenge urine test (challenge with a chelator)? How many have

chelated their children? How many have read the autism or ADHD

biomedical literature even though their child is nowhere near

autistic or ADHD? There will of course be different issues for

different kids and different disorders, but if the biomedical

protocol involves evaluation and testing first and only then treating

and healing as indicated by the tests, then it can be as individual

as you need it to be. And maybe that's why some children respond

immediately to ProEFA, and others don't. Other unresolved issues get

in the way of the ProEFA, or detract from what it could be doing.

Okay, I hope that everyone's holidays were and will be fantastic. I

just really want to talk about all this, because it could actually

hold out hope for my beloved Lulu (who turned 2 years old yesterday

and began making cow, rooster and cat sounds for the first time --

yippee! Yes, they all sound kind of the same, but we and she know

they're not).

Lots of love to you and all your children,

Theresa

-- In , " deverelementary "

<kearneysix@a...> wrote:

>

>

> The implications of these findings extend well beyond thimerosal

and

> autism. Reduced antioxidant defense may characterize a group of

> individuals who are demonstrably more sensitive to the effects of a

> range of toxic chemical exposures, and shed light on increasing

rates

> of related learning and behavioral disorders.

>

>

> My son was a participant in the follow up studies, not autistic,

yet

> had apraxia, major sensory issues, coordination problems, the

> nutritional interventions he was given/supplements, reversed all of

> his issues, this is a fact.So this goes way beyond just autism, a

> friends child with CAPD,ADD was also a participant and he too is

now

> without any diagnosis of CAPD/ADD.

>

> Colleen

> >

> > http://www.ewg.org/reports/autism/execsumm.php

> >

> >

> >

[Guest guest]

I would also be very appreciative of this information ,I finally have a ped that

is " somewhat " openminded perhaps this will persuade her.All four of my kids are

on the gfcfsf diet and take NN compete.2 have ASD,1 probably add,and the baby

at 8 months was showing some asd traits and had little verbal skills.3 of the

are doing really ,really well.The baby now 19 months talks in 3 and 4 word

sentences and is sleeping much better,he also has had no vaccs since 4 months of

age,If I can find a safe way to get him immunized

I will ,until then,no way.This was one reason I fired my last ped,she had no

time to discuss biomedical issues but would spend 20 minutes trying to pressure

me into vaccinating my baby.On my last visit with her I said sure,give him the

shots and since they are so safe sign that you will be financially responsible

for him if he becomes autistic,funny,she did not want to sign it

Also a Theresa

[ ] Re:EWG Report ---- Autism report news release

12.13.04

Hello Everyone and Hello Colleen,

This EWG report is astonishing and I am surprised that the list

didn't go crazy over this very important finding which could affect

all of us on this list. My impression is that we need a serious

discussion of the report and its possible bearing on apraxia.

The report -- like all the ground-breaking research -- centers on

autism, but it states several times that it relates to other

neurodevelopmental disorders. It sounds like it could provide THE KEY

for the vast majority of our apraxic children. It explains so much --

the reason why so many kids with all kinds of neurodevelopmental

learning disorders live in the same neighborhoods (for example, why

three of the four children I know who were adopted from the same

orphanage in Bethlehem all have neurodevelopmental learning

disorders), why there has been such an explosion of

neurodevelopmental learning disorders over the past decades, why so

many neurodevelopmental learning disorders overlap and are co-morbid,

why non-autistic children have been helped and sometimes cured

through biomedical protocols designed for autistic children.

I need no convincing -- I believe that in a great number of cases,

the various disorders (the autism spectrum disorders, ADD/ADHD,

apraxia, sensory issues, some forms of cerebral palsy, some forms of

brain damage, some forms of microcephaly, etc.) are related and that

the glutathione/heavy metal connection is central. I don't need to

wait for the double-blind clinical trials to go out now and try this

new glutathione and methylcobalamin S. Jill protocol.

When Colleen says that her son was cured of his apraxia, sensory

issues and coordination problems, that another child was cured of

CAPD and ADD, we should all be clamoring to hear where we can sign

up. This is INCREDIBLE, and if you read the report, all the " Aha!

Yes! This is IT! " bells will go off in your head. At least for a

great number of children suffering from these neurodevelopmental

disorders. No, not all, but just very possibly a majority.

Colleen, is there any way that we can get from you the protocol that

Dr. followed? I want to know just what she did in the follow-

up study your son paricipated in. There is a little bit of

information in the last appendix of the 2004 second edition of

Children with Starving Brains by McCandless, but not

really enough to show to a doctor and say " Do this, please. " I'm

particularly interested in getting the protocol because I live

in the Netherlands now and have to basically " teach " a willing doctor

here what to do.

How many parents on this list have followed a DAN! protocol for their

non-autistic child? How many have had their child tested for heavy

metal poisoning -- with a hair strand test AND a pre- and post-

challenge urine test (challenge with a chelator)? How many have

chelated their children? How many have read the autism or ADHD

biomedical literature even though their child is nowhere near

autistic or ADHD? There will of course be different issues for

different kids and different disorders, but if the biomedical

protocol involves evaluation and testing first and only then treating

and healing as indicated by the tests, then it can be as individual

as you need it to be. And maybe that's why some children respond

immediately to ProEFA, and others don't. Other unresolved issues get

in the way of the ProEFA, or detract from what it could be doing.

Okay, I hope that everyone's holidays were and will be fantastic. I

just really want to talk about all this, because it could actually

hold out hope for my beloved Lulu (who turned 2 years old yesterday

and began making cow, rooster and cat sounds for the first time --

yippee! Yes, they all sound kind of the same, but we and she know

they're not).

Lots of love to you and all your children,

Theresa

-- In , " deverelementary "

<kearneysix@a...> wrote:

>

>

> The implications of these findings extend well beyond thimerosal

and

> autism. Reduced antioxidant defense may characterize a group of

> individuals who are demonstrably more sensitive to the effects of a

> range of toxic chemical exposures, and shed light on increasing

rates

> of related learning and behavioral disorders.

>

>

> My son was a participant in the follow up studies, not autistic,

yet

> had apraxia, major sensory issues, coordination problems, the

> nutritional interventions he was given/supplements, reversed all of

> his issues, this is a fact.So this goes way beyond just autism, a

> friends child with CAPD,ADD was also a participant and he too is

now

> without any diagnosis of CAPD/ADD.

>

> Colleen

> >

> > http://www.ewg.org/reports/autism/execsumm.php

> >

> >

> >

[Guest guest]

>

> Colleen, is there any way that we can get from you the protocol

that

> Dr. followed? I want to know just what she did in the follow-

> up study your son paricipated in. There is a little bit of

> information in the last appendix of the 2004 second edition of

> Children with Starving Brains by McCandless, but not

> really enough to show to a doctor and say " Do this, please. " I'm

> particularly interested in getting the protocol because I

live

> in the Netherlands now and have to basically " teach " a willing

doctor

> here what to do.

Theresa,

No problem, strict gf/cf/soyfree/preservative,egg,chocolate,corn,

corn syrup,peanut,sugar, color/dye free,fluoride free for one month,

second month begin nutritional supplements:

Nuthera 2 capsules a day one am one pm

TMG one am one pm

Folinic Acid(folinic NOT folic) two capsules a day one am one pm

The above are the supplements to drastically increase glutathione

(MAJOR anti-oxidant in the body, boys have less than females, illness

lowers it, so do environmental assaults ie,vaccines, car

exhaust,pesticides in foods, etc)

Diet wise second month challenge foods, sugar in excess for a day,

watch for reactions,reactions may last as long as 3 days, ANY change

is significant, looser stools,firmer stools, sleepy,hyper, red

eyes,red ears,behavior.

If no reaction challenge another food, say dyes, give kool aid in

excess, the mix you make with regular sugar one food item you already

challenged and had no reaction to.Wait 3 days, then do say,

preservatives, let them eat a hot dog that has preservatives(major

offender BHT, MSG)

Once you are aware of what causes issues, you just don't let them eat

it, meanwhile still gf/cf. As time goes on and body/heatlh is

improving, a little skittles here or there, or starburst is ok.

Major to the improvements is getting bowel movements daily, we were

told to use:(42 pound 6 year old)

Magnessium citrate one teaspoon daily (1/2 am 1/2 pm)

Zinc one teaspoon daily at bedtime (away from food, doesn't absorb

well w/food in stomach.

ProEFA 3 caps daily, on it's own

Month 3 we added methyl b 12 every other day, steady

balance/coordintaion improvements the longer we gave this beynd the 3

month study dictates.

N-AC was added to the mix in month 4 huge nurturing/affctionate

skills came with this one, reading ability skyrocketed, major

handwriting improvements/neatness/speed.Teacher told me at last

parent teacher conference (November, he is way ahead of the other

children (mainstream 1st grade, no IEP, no speech, no OT/PT)in all

areas, reading, spelling, math. I was speechless, but had noticed it

all at home with his ability to read his sisters pleasure books,

Lemony snickets series.)

Teacher said, he is ALWAYS focused and in tune, she said she wished

the whole class would do what he is doing.

Turn the clock back one year ago, he was crying in class,unable to

pronounce letters, or sounds of them, chewing his clothing,(came home

many a day with missing shirt buttons), sppech therapy twice a weeek,

one step foward two backwards. no consistancy with progress, same as

for PT/OT, one day ability was there, the next day gone. Consistantly

inconsistant.

We are still in the study, and as mentioned above it is what we are

doing, now in the process, of cutting down on the m b12, to see what

his right fit is, it is not a deficiency of b12 that drives this, it

is the inablity of the body to use the form it has, so more of a

dependancy of mb12, so we are trying to find his lowest dose of need,

and on we go..

Just a point of note, DIET is major here, you really can't do one

without the other, it is a sensitivity issue, any inflammation

impedes the nutritional uptake of what the child needs, and the most

common offenders are gluten/casein.The immune system is totally

regulated in the gut, it is where glutathione is formed with the

liver/intestines/stomach being the sources of this process.

The things she measured that would indicate reduced ability to detox

would be a low methionine level, low homocysteine, low cysteine

level.these numbers were accross the board with AD/ADHD, autism, CAPD

the numbers were not any different among the children, the only thing

different was the childs response to these numbers, they are all

affected differently.

She did a thiol profile, which measures other detox pathways in the

body, I believe there are six pathways she tested for, but the most

prevalent, (which is according to researchers, about 40% of

caucasions)is the MTHFR gene, some of the others she tested came in

around 20-30% some only 10-15% so that is what McCAndeles mentions is

the appendix of children with starving brains, most common and

TMG/Folinic/Mb12 does the trick.Unfortunately Jill lab is the

only lab in the country doing the blood testing, at this point.

Being homozygous for MTHFR you have a 60 % reduced ability to detox

toxins, heterozygous a 30% reduced ability, we are heterozygous all 6

of us at our household.And my son (I have 3 girls also) is the only

one that has had a problem, I do see some things in my girls but not

affecting them cognitively, and my youngest wont be getting anymore

vaccines, just to be on the safe side.

I have sent 9 people to get tested(not related to me in anyway) and

all 9 are positive, and they all have kids that have various issues,

CAPD, AD/ADHD, speech disorders, PDD, autism.And their kids are all

positive also, so pretty statistically significant if you are a

numbers person.

One thing to note, a starting point bllod work wise, my son's blood

was the only one from our house that was sent to Jill because

of his cognitive issues, doctor sent our bloods to regular lab, for

Methylenetetrahydrofolate Reductse (MTHFR)

Homocysteine,

serum b12,

rbc folate

b6

As far as brand of supplements for the study, they were all Kirkmans,

and Proefa was Nordic Naturals. Not pushing any Kirkmans products but

was part of the study for research purposes, no variables.

So, Theresa, that is most of it in a LONG nut shell, I hope this

helps you get to where we are, and you are way ahead of the game, she

is sooooo young, the younger the better.Good luck, and e-mail me when

ever you have any questions, be so glad to help if I can, the results

are amazing to say the least.

Colleen

[Guest guest]

Colleen,

Thank you for posting this,I have been trying to find this specific info for

awhile.May I ask you a few more questions?What did you use to replace the

sugar?Are multi-vits ok in month 1?Like polyvisol.Also if you were or are doing

yeast/and or viral treatments do you stop?And lastly do you have to get a script

for the b-12?Where is it recommended to obtain it?Did you also use the lipo/gsh

everyone is so pleased with.

Thank you so much

also a theresa but not same you replied to

mom to 5

2 asd

[ ] Re:EWG Report ---- Autism report news release

12.13.04

>

> Colleen, is there any way that we can get from you the protocol

that

> Dr. followed? I want to know just what she did in the follow-

> up study your son paricipated in. There is a little bit of

> information in the last appendix of the 2004 second edition of

> Children with Starving Brains by McCandless, but not

> really enough to show to a doctor and say " Do this, please. " I'm

> particularly interested in getting the protocol because I

live

> in the Netherlands now and have to basically " teach " a willing

doctor

> here what to do.

Theresa,

No problem, strict gf/cf/soyfree/preservative,egg,chocolate,corn,

corn syrup,peanut,sugar, color/dye free,fluoride free for one month,

second month begin nutritional supplements:

Nuthera 2 capsules a day one am one pm

TMG one am one pm

Folinic Acid(folinic NOT folic) two capsules a day one am one pm

The above are the supplements to drastically increase glutathione

(MAJOR anti-oxidant in the body, boys have less than females, illness

lowers it, so do environmental assaults ie,vaccines, car

exhaust,pesticides in foods, etc)

Diet wise second month challenge foods, sugar in excess for a day,

watch for reactions,reactions may last as long as 3 days, ANY change

is significant, looser stools,firmer stools, sleepy,hyper, red

eyes,red ears,behavior.

If no reaction challenge another food, say dyes, give kool aid in

excess, the mix you make with regular sugar one food item you already

challenged and had no reaction to.Wait 3 days, then do say,

preservatives, let them eat a hot dog that has preservatives(major

offender BHT, MSG)

Once you are aware of what causes issues, you just don't let them eat

it, meanwhile still gf/cf. As time goes on and body/heatlh is

improving, a little skittles here or there, or starburst is ok.

Major to the improvements is getting bowel movements daily, we were

told to use:(42 pound 6 year old)

Magnessium citrate one teaspoon daily (1/2 am 1/2 pm)

Zinc one teaspoon daily at bedtime (away from food, doesn't absorb

well w/food in stomach.

ProEFA 3 caps daily, on it's own

Month 3 we added methyl b 12 every other day, steady

balance/coordintaion improvements the longer we gave this beynd the 3

month study dictates.

N-AC was added to the mix in month 4 huge nurturing/affctionate

skills came with this one, reading ability skyrocketed, major

handwriting improvements/neatness/speed.Teacher told me at last

parent teacher conference (November, he is way ahead of the other

children (mainstream 1st grade, no IEP, no speech, no OT/PT)in all

areas, reading, spelling, math. I was speechless, but had noticed it

all at home with his ability to read his sisters pleasure books,

Lemony snickets series.)

Teacher said, he is ALWAYS focused and in tune, she said she wished

the whole class would do what he is doing.

Turn the clock back one year ago, he was crying in class,unable to

pronounce letters, or sounds of them, chewing his clothing,(came home

many a day with missing shirt buttons), sppech therapy twice a weeek,

one step foward two backwards. no consistancy with progress, same as

for PT/OT, one day ability was there, the next day gone. Consistantly

inconsistant.

We are still in the study, and as mentioned above it is what we are

doing, now in the process, of cutting down on the m b12, to see what

his right fit is, it is not a deficiency of b12 that drives this, it

is the inablity of the body to use the form it has, so more of a

dependancy of mb12, so we are trying to find his lowest dose of need,

and on we go..

Just a point of note, DIET is major here, you really can't do one

without the other, it is a sensitivity issue, any inflammation

impedes the nutritional uptake of what the child needs, and the most

common offenders are gluten/casein.The immune system is totally

regulated in the gut, it is where glutathione is formed with the

liver/intestines/stomach being the sources of this process.

The things she measured that would indicate reduced ability to detox

would be a low methionine level, low homocysteine, low cysteine

level.these numbers were accross the board with AD/ADHD, autism, CAPD

the numbers were not any different among the children, the only thing

different was the childs response to these numbers, they are all

affected differently.

She did a thiol profile, which measures other detox pathways in the

body, I believe there are six pathways she tested for, but the most

prevalent, (which is according to researchers, about 40% of

caucasions)is the MTHFR gene, some of the others she tested came in

around 20-30% some only 10-15% so that is what McCAndeles mentions is

the appendix of children with starving brains, most common and

TMG/Folinic/Mb12 does the trick.Unfortunately Jill lab is the

only lab in the country doing the blood testing, at this point.

Being homozygous for MTHFR you have a 60 % reduced ability to detox

toxins, heterozygous a 30% reduced ability, we are heterozygous all 6

of us at our household.And my son (I have 3 girls also) is the only

one that has had a problem, I do see some things in my girls but not

affecting them cognitively, and my youngest wont be getting anymore

vaccines, just to be on the safe side.

I have sent 9 people to get tested(not related to me in anyway) and

all 9 are positive, and they all have kids that have various issues,

CAPD, AD/ADHD, speech disorders, PDD, autism.And their kids are all

positive also, so pretty statistically significant if you are a

numbers person.

One thing to note, a starting point bllod work wise, my son's blood

was the only one from our house that was sent to Jill because

of his cognitive issues, doctor sent our bloods to regular lab, for

Methylenetetrahydrofolate Reductse (MTHFR)

Homocysteine,

serum b12,

rbc folate

b6

As far as brand of supplements for the study, they were all Kirkmans,

and Proefa was Nordic Naturals. Not pushing any Kirkmans products but

was part of the study for research purposes, no variables.

So, Theresa, that is most of it in a LONG nut shell, I hope this

helps you get to where we are, and you are way ahead of the game, she

is sooooo young, the younger the better.Good luck, and e-mail me when

ever you have any questions, be so glad to help if I can, the results

are amazing to say the least.

Colleen

[Guest guest]

Colleen,

Thanks so much for posting this information! Did you supplement methyl b12

orally or through injections?

Dina

In a message dated 12/28/2004 1:24:03 PM Eastern Standard Time,

writes:

Month 3 we added methyl b 12 every other day

[Guest guest]

Colleen and Everyone,

Thanks so fantastically much for this! I'm going to digest it all and will

probably ask some clarifying questions based on what I know from the Children

with Starving Brains book, which basically sets out what Binstock told me

was a " middle of the road " DAN! protocol. I know, for example, that testing and

evaluation is important, so I will want to know if the tests you included hee

are the only tests that were run, rather than all the standard ones first, etc.

As a frist reaction, how did you implement this diet? Wow, I have tons of work

to do just to get started on it! I've been figuring out how to implement the

GF/CF, but to add soy, egg and corn to the banned list takes away many of my

substitute foods! Anyone know of an asian cookbook for kids? But I am in jest

-- we will figure it all out, not to worry -- I'm just venting in a vaguely

humorous way!

Do you know if there is a way for me to get more information on the S Jill

study you are part of, other articles by her, contact information for her? I

would very much like the docotor (and associated laboratory - the European

Laboratory of Nutrients) we have contacted here and who has attended several

DAN! conferences in the US to contact her about this. Perhaps she can give us

more information for our docotors here on the protocol for the study you are

with. Can you tell me or point me to infomration on what the working hypothesis

of the study you're with is, why it included non-autistic children, etc? Is this

a published clinical trial or something? If so, there is probably a website

that announces it. Anything you can give me would be great as I must get most

of my info through the internet.

I am still in a transition period and will hopefully be moving today into our

temporary housing today (3 months) as we await our furniture's arrival by ship.

It MAY take me several days to get online again, I just don't know. Please bear

with my silence if I don't respond immediately.

I am very excited about this and feel very strongly that it could help us. I

hope that others will try it, too.

Much love and peace to all of you and Happy New Year,

Theresa

Colleen, is

Message: 6

Date: Tue, 28 Dec 2004 05:56:28 -0000

From: " deverelementary "

Subject: Re:EWG Report ---- Autism report news release 12.13.04

Theresa,

No problem, strict gf/cf/soyfree/preservative,egg,chocolate,corn,

corn syrup,peanut,sugar, color/dye free,fluoride free for one month,

second month begin nutritional supplements:

Nuthera 2 capsules a day one am one pm

TMG one am one pm

Folinic Acid(folinic NOT folic) two capsules a day one am one pm

The above are the supplements to drastically increase glutathione

(MAJOR anti-oxidant in the body, boys have less than females, illness

lowers it, so do environmental assaults ie,vaccines, car

exhaust,pesticides in foods, etc)

Diet wise second month challenge foods, sugar in excess for a day,

watch for reactions,reactions may last as long as 3 days, ANY change

is significant, looser stools,firmer stools, sleepy,hyper, red

eyes,red ears,behavior.

If no reaction challenge another food, say dyes, give kool aid in

excess, the mix you make with regular sugar one food item you already

challenged and had no reaction to.Wait 3 days, then do say,

preservatives, let them eat a hot dog that has preservatives(major

offender BHT, MSG)

Once you are aware of what causes issues, you just don't let them eat

it, meanwhile still gf/cf. As time goes on and body/heatlh is

improving, a little skittles here or there, or starburst is ok.

Major to the improvements is getting bowel movements daily, we were

told to use:(42 pound 6 year old)

Magnessium citrate one teaspoon daily (1/2 am 1/2 pm)

Zinc one teaspoon daily at bedtime (away from food, doesn't absorb

well w/food in stomach.

ProEFA 3 caps daily, on it's own

Month 3 we added methyl b 12 every other day, steady

balance/coordintaion improvements the longer we gave this beynd the 3

month study dictates.

N-AC was added to the mix in month 4 huge nurturing/affctionate

skills came with this one, reading ability skyrocketed, major

handwriting improvements/neatness/speed.Teacher told me at last

parent teacher conference (November, he is way ahead of the other

children (mainstream 1st grade, no IEP, no speech, no OT/PT)in all

areas, reading, spelling, math. I was speechless, but had noticed it

all at home with his ability to read his sisters pleasure books,

Lemony snickets series.)

Teacher said, he is ALWAYS focused and in tune, she said she wished

the whole class would do what he is doing.

Turn the clock back one year ago, he was crying in class,unable to

pronounce letters, or sounds of them, chewing his clothing,(came home

many a day with missing shirt buttons), sppech therapy twice a weeek,

one step foward two backwards. no consistancy with progress, same as

for PT/OT, one day ability was there, the next day gone. Consistantly

inconsistant.

We are still in the study, and as mentioned above it is what we are

doing, now in the process, of cutting down on the m b12, to see what

his right fit is, it is not a deficiency of b12 that drives this, it

is the inablity of the body to use the form it has, so more of a

dependancy of mb12, so we are trying to find his lowest dose of need,

and on we go..

Just a point of note, DIET is major here, you really can't do one

without the other, it is a sensitivity issue, any inflammation

impedes the nutritional uptake of what the child needs, and the most

common offenders are gluten/casein.The immune system is totally

regulated in the gut, it is where glutathione is formed with the

liver/intestines/stomach being the sources of this process.

The things she measured that would indicate reduced ability to detox

would be a low methionine level, low homocysteine, low cysteine

level.these numbers were accross the board with AD/ADHD, autism, CAPD

the numbers were not any different among the children, the only thing

different was the childs response to these numbers, they are all

affected differently.

She did a thiol profile, which measures other detox pathways in the

body, I believe there are six pathways she tested for, but the most

prevalent, (which is according to researchers, about 40% of

caucasions)is the MTHFR gene, some of the others she tested came in

around 20-30% some only 10-15% so that is what McCAndeles mentions is

the appendix of children with starving brains, most common and

TMG/Folinic/Mb12 does the trick.Unfortunately Jill lab is the

only lab in the country doing the blood testing, at this point.

Being homozygous for MTHFR you have a 60 % reduced ability to detox

toxins, heterozygous a 30% reduced ability, we are heterozygous all 6

of us at our household.And my son (I have 3 girls also) is the only

one that has had a problem, I do see some things in my girls but not

affecting them cognitively, and my youngest wont be getting anymore

vaccines, just to be on the safe side.

I have sent 9 people to get tested(not related to me in anyway) and

all 9 are positive, and they all have kids that have various issues,

CAPD, AD/ADHD, speech disorders, PDD, autism.And their kids are all

positive also, so pretty statistically significant if you are a

numbers person.

One thing to note, a starting point bllod work wise, my son's blood

was the only one from our house that was sent to Jill because

of his cognitive issues, doctor sent our bloods to regular lab, for

Methylenetetrahydrofolate Reductse (MTHFR)

Homocysteine,

serum b12,

rbc folate

b6

As far as brand of supplements for the study, they were all Kirkmans,

and Proefa was Nordic Naturals. Not pushing any Kirkmans products but

was part of the study for research purposes, no variables.

So, Theresa, that is most of it in a LONG nut shell, I hope this

helps you get to where we are, and you are way ahead of the game, she

is sooooo young, the younger the better.Good luck, and e-mail me when

ever you have any questions, be so glad to help if I can, the results

are amazing to say the least.

Colleen

[Guest guest]

Theresa,

We never had viral issues,Thank god, nor yeast, if we did have yeast

it was a minor player, others however have major yeast/viral issues,

I know of one child that has a latent rubella virus in her brain(humm

how did that get there, yes she had all her vaccines on time)As for

sugar replacements, pure maple syrup*the expensive stuff), or honey

is ok,I also used pure maple sugar, sold at Whole foods, my son

is/wasn't a sugarholic so I used it infrequently. I would guess any

multi vit is ok, anything w/o sugar or dyes,we used no multi at all

the first month, but we had to follow the trial. and definitelly NO

FLOURIDE< of any shape or form, not in drops or toothpaste. Remember

flouride is a metal, and if someone can't get rid of metals, (like my

son) fluoride will go right to to bones, (fluorosis).Our (remember it

is methyl b12 not hydro or cynocobalamin) there is a difference. )

was an injection as part of the protocol. We got ours from ped

allergist/immunologist, And it is a script item, however many others

have had really good results with the sublingual mb12, Thorne

research has a TMG/Folinic/mb12 supplement can be ordered on line,

also kirkmans carries a product also. As far as the lipo/gsh, we

never needed it, his glutathione numbers went up with the

tmg/folinic/mb12, so I can't say one way or the other, but others use

it and see really good things with it.Hope this helps

Colleen

> >

> > Colleen, is there any way that we can get from you the

protocol

> that

> > Dr. followed? I want to know just what she did in the

follow-

> > up study your son paricipated in. There is a little bit of

> > information in the last appendix of the 2004 second edition of

> > Children with Starving Brains by McCandless, but not

> > really enough to show to a doctor and say " Do this, please. "

I'm

> > particularly interested in getting the protocol because I

> live

> > in the Netherlands now and have to basically " teach " a willing

> doctor

> > here what to do.

>

>

> Theresa,

>

> No problem, strict gf/cf/soyfree/preservative,egg,chocolate,corn,

> corn syrup,peanut,sugar, color/dye free,fluoride free for one

month,

> second month begin nutritional supplements:

> Nuthera 2 capsules a day one am one pm

> TMG one am one pm

> Folinic Acid(folinic NOT folic) two capsules a day one am one pm

> The above are the supplements to drastically increase glutathione

> (MAJOR anti-oxidant in the body, boys have less than females,

illness

> lowers it, so do environmental assaults ie,vaccines, car

> exhaust,pesticides in foods, etc)

>

> Diet wise second month challenge foods, sugar in excess for a

day,

> watch for reactions,reactions may last as long as 3 days, ANY

change

> is significant, looser stools,firmer stools, sleepy,hyper, red

> eyes,red ears,behavior.

>

> If no reaction challenge another food, say dyes, give kool aid in

> excess, the mix you make with regular sugar one food item you

already

> challenged and had no reaction to.Wait 3 days, then do say,

> preservatives, let them eat a hot dog that has preservatives

(major

> offender BHT, MSG)

>

> Once you are aware of what causes issues, you just don't let them

eat

> it, meanwhile still gf/cf. As time goes on and body/heatlh is

> improving, a little skittles here or there, or starburst is ok.

> Major to the improvements is getting bowel movements daily, we

were

> told to use:(42 pound 6 year old)

> Magnessium citrate one teaspoon daily (1/2 am 1/2 pm)

> Zinc one teaspoon daily at bedtime (away from food, doesn't

absorb

> well w/food in stomach.

> ProEFA 3 caps daily, on it's own

>

> Month 3 we added methyl b 12 every other day, steady

> balance/coordintaion improvements the longer we gave this beynd

the 3

> month study dictates.

> N-AC was added to the mix in month 4 huge nurturing/affctionate

> skills came with this one, reading ability skyrocketed, major

> handwriting improvements/neatness/speed.Teacher told me at last

> parent teacher conference (November, he is way ahead of the other

> children (mainstream 1st grade, no IEP, no speech, no OT/PT)in

all

> areas, reading, spelling, math. I was speechless, but had noticed

it

> all at home with his ability to read his sisters pleasure books,

> Lemony snickets series.)

>

> Teacher said, he is ALWAYS focused and in tune, she said she

wished

> the whole class would do what he is doing.

> Turn the clock back one year ago, he was crying in class,unable

to

> pronounce letters, or sounds of them, chewing his clothing,(came

home

> many a day with missing shirt buttons), sppech therapy twice a

weeek,

> one step foward two backwards. no consistancy with progress, same

as

> for PT/OT, one day ability was there, the next day gone.

Consistantly

> inconsistant.

>

> We are still in the study, and as mentioned above it is what we

are

> doing, now in the process, of cutting down on the m b12, to see

what

> his right fit is, it is not a deficiency of b12 that drives this,

it

> is the inablity of the body to use the form it has, so more of a

> dependancy of mb12, so we are trying to find his lowest dose of

need,

> and on we go..

>

> Just a point of note, DIET is major here, you really can't do one

> without the other, it is a sensitivity issue, any inflammation

> impedes the nutritional uptake of what the child needs, and the

most

> common offenders are gluten/casein.The immune system is totally

> regulated in the gut, it is where glutathione is formed with the

> liver/intestines/stomach being the sources of this process.

>

> The things she measured that would indicate reduced ability to

detox

> would be a low methionine level, low homocysteine, low cysteine

> level.these numbers were accross the board with AD/ADHD, autism,

CAPD

> the numbers were not any different among the children, the only

thing

> different was the childs response to these numbers, they are all

> affected differently.

>

> She did a thiol profile, which measures other detox pathways in

the

> body, I believe there are six pathways she tested for, but the

most

> prevalent, (which is according to researchers, about 40% of

> caucasions)is the MTHFR gene, some of the others she tested came

in

> around 20-30% some only 10-15% so that is what McCAndeles

mentions is

> the appendix of children with starving brains, most common and

> TMG/Folinic/Mb12 does the trick.Unfortunately Jill lab is

the

> only lab in the country doing the blood testing, at this point.

>

> Being homozygous for MTHFR you have a 60 % reduced ability to

detox

> toxins, heterozygous a 30% reduced ability, we are heterozygous

all 6

> of us at our household.And my son (I have 3 girls also) is the

only

> one that has had a problem, I do see some things in my girls but

not

> affecting them cognitively, and my youngest wont be getting

anymore

> vaccines, just to be on the safe side.

>

> I have sent 9 people to get tested(not related to me in anyway)

and

> all 9 are positive, and they all have kids that have various

issues,

> CAPD, AD/ADHD, speech disorders, PDD, autism.And their kids are

all

> positive also, so pretty statistically significant if you are a

> numbers person.

>

> One thing to note, a starting point bllod work wise, my son's

blood

> was the only one from our house that was sent to Jill

because

> of his cognitive issues, doctor sent our bloods to regular lab,

for

>

> Methylenetetrahydrofolate Reductse (MTHFR)

> Homocysteine,

> serum b12,

> rbc folate

> b6

>

>

> As far as brand of supplements for the study, they were all

Kirkmans,

> and Proefa was Nordic Naturals. Not pushing any Kirkmans products

but

> was part of the study for research purposes, no variables.

>

> So, Theresa, that is most of it in a LONG nut shell, I hope this

> helps you get to where we are, and you are way ahead of the game,

she

> is sooooo young, the younger the better.Good luck, and e-mail me

when

> ever you have any questions, be so glad to help if I can, the

results

> are amazing to say the least.

>

> Colleen

>

>

>

>

>

>

>

>

>

>

[Guest guest]

Theresa:

I agree with you, the EWG report seems remarkable. How we can find

out more about the protocol that Dr. followed.

It would be completely amazing, not short of a miracle if our

children could be cured of apraxia...

Does anyone in our group have more information regarding this EWG

report and the protocol? I'm up for discussion on this important

issue.

G.-- Do you have any insight regarding the EWG Report and the

protocol? What are your thoughts?

Thanks,

,

[Guest guest]

To All:

This is the journal article as it appeared in the Journal of

Neurotoxicology, study by Jill et al.

1 of 18

NeuroToxicology

Volume 26, Issue 1 , January 2005, Pages 1-8

This Document

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doi:10.1016/j.neuro.2004.07.012

Copyright © 2004 Elsevier Inc. All rights reserved.

Thimerosal Neurotoxicity is Associated with Glutathione Depletion:

Protection with Glutathione Precursors

S.J. 1, , , Slikker III2, Stepan Melnyk1,

New2, Marta Pogribna2 and Stefanie Jernigan1

1Department of Pediatrics, University of Arkansas for Medical

Sciences and Arkansas Children's Hospital Research Institute, Little

Rock, AR 72202, USA

2Division of Biochemical Toxicology, National Center for

Toxicological Research, Jefferson, AR 72079, USA

Received 24 May 2004; accepted 28 July 2004. Available online 29

September 2004.

Abstract

Thimerosol is an antiseptic containing 49.5% ethyl mercury that has

been used for years as a preservative in many infant vaccines and in

flu vaccines. Environmental methyl mercury has been shown to be

highly neurotoxic, especially to the developing brain. Because

mercury has a high affinity for thiol (sulfhydryl (SH)) groups, the

thiol-containing antioxidant, glutathione (GSH), provides the major

intracellular defense against mercury-induced neurotoxicity. Cultured

neuroblastoma cells were found to have lower levels of GSH and

increased sensitivity to thimerosol toxicity compared to glioblastoma

cells that have higher basal levels of intracellular GSH. Thimerosal-

induced cytotoxicity was associated with depletion of intracellular

GSH in both cell lines. Pretreatment with 100 & #956;M glutathione ethyl

ester or N-acetylcysteine (NAC), but not methionine, resulted in a

significant increase in intracellular GSH in both cell types.

Further, pretreatment of the cells with glutathione ethyl ester or

NAC prevented cytotoxicity with exposure to 15 & #956;M Thimerosal.

Although Thimerosal has been recently removed from most children's

vaccines, it is still present in flu vaccines given to pregnant

women, the elderly, and to children in developing countries. The

potential protective effect of GSH or NAC against mercury toxicity

warrants further research as possible adjunct therapy to individuals

still receiving Thimerosal-containing vaccinations.

Keywords: Thimerosal; Neurotoxicity; Glutathione; N-acetylcysteine

Article Outline

INTRODUCTION

MATERIALS AND METHODS

Materials

Cell Culture

Cell Viability Assays

Nutrient Supplementation Studies

HPLC Sample Preparation

HPLC with Coulometric Electrochemical Detection

Statistics

RESULTS

Dose–Response Characteristics of Thimerosal in Glioblastoma and

Neuroblastoma Cells

Neuroprotective Effect of N-Acetylcysteine, Cystine, and Glutathione

Ethyl Ester

Glioblastoma Cells

Neuroblastoma Cells

Glutathione Depletion with Thimerosal Exposure: Preservation of

Intracellular GSH with Nutritional Supplementation

DISCUSSION

References

INTRODUCTION

Thimerosal (sodium ethylmercurithiosalicylate) was developed by Eli

Lilly in the 1930s as a effective bacteriostatic and fungistatic

preservative and has been widely used in multidose vials of vaccines

and in ophthalmic, otic, nasal, and topical products. Until the

removal of Thimerosal from most pediatric vaccines in 2001, the

largest human exposure in the US ( & #956;g/kg body weight) was in children

under 18 months of age undergoing routine childhood immunization

schedules. Prior to 2001, a child may have received a cumulative dose

of over 200 & #956;g/kg in the first 18 months of life (Ball et al., 2001).

Although the neurotoxicity of methyl mercury has been relatively well

studied, limited information is available on the relative

neurodevelopmental toxicity of ethylmercury, the mercury metabolite

of Thimerosal. Based on the known toxicity of methylmercury, the

cumulative ethylmercury exposure to US pediatric populations in

Thimerosal-containing vaccinations was re-examined in 1999 and found

to exceed EPA recommended guidelines (Ball et al., 2001). Following

recommendations by the American Academy of Pediatrics and the US

Public Health Service (Public Health Service, 1999), Thimerosal was

subsequently removed as a preservative from most children's vaccines

in the US. However, influenza vaccines and Rho D immunoglobulin shots

containing Thimerosal are still recommended to pregnant women, and

many vaccines given to children in developing countries still contain

Thimerosal. The present study was undertaken to better understand the

mechanisms underlying Thimerosal toxicity to neurons and astrocytes,

the primary CNS targets for organic mercury (Sanfeliu et al., 2001).

A better understanding of the neurotoxic mechanism is a necessary

prerequisite for the design of intervention strategies for prevention

and for the identification of genetic variants that could increase

sensitivity to Thimerosal.

Previous mechanistic studies of methylmercury toxicity in astrocytes

and neurons have implicated reactive oxygen species (ROS) and

depletion of intracellular glutathione as major contributors to

mercury-induced cytotoxicity (Sanfeliu et al., 2001). Organic mercury

has a high affinity for the thiol (SH) group on glutathione, a

tripeptide composed of cysteine, glutamate, and glycine (Sanfeliu et

al., 2003). The cysteine moiety of glutathione carries the active

thiol group that binds and detoxifies a variety of heavy metals,

including organic and inorganic mercury. Normally, the intracellular

concentration of glutathione is extremely high, in the mM range

(Meister, 1995); however, with depletion of this essential

antioxidant, excess free mercury is available to bind to cysteine

thiol groups present in essential cellular proteins, leading to

functional inactivation and cytotoxicity.

The synthesis of glutathione in the brain is unique in that brain

cells do not express cystathionine gamma lyase, an enzyme in the

transsulfuration pathway involved in glutathione synthesis (Awata et

al., 1995). As a result, brain cells cannot synthesize cysteine, the

rate limiting amino acid for glutathione synthesis. Thus, the brain

is dependent on the liver to synthesize and export cysteine for

uptake and utilization by astrocytes and neurons for adequate

glutathione synthesis (Lu, 1998). In contrast to the brain, the liver

expresses the complete transsulfuration pathway from methionine to

cysteine and glutathione as diagrammed in Fig. 1. Glutathione

synthesized in the liver is exported to the plasma where it is

immediately degraded to cysteinylglycine and cysteine (Lu, 1998).

Cysteine is converted to cystine in the oxidizing environment of the

plasma and subsequently transported to the brain for intracellular

glutathione synthesis (Fig. 2).

(29K)

Fig. 1. Pathways of glutathione synthesis. The liver expresses the

complete pathway from methionine through homocysteine and cysteine to

glutathione. Astrocytes and neurons do not express the enzyme

cystathionine lyase and therefore are unable to synthesize cysteine.

As a result, astrocytes and neurons are dependent on plasma cysteine

derived primarily from the liver to synthesize glutathione.

(6K)

Fig. 2. Interaction between astrocytes and neurons for glutathione

synthesis. Glutathione exported from the liver is hydrolyzed to

cysteinylglycine and cysteine. In the plasma, cysteine is oxidized to

cystine and transported across the BBB and taken up by astrocytes and

used to synthesize glutathione. Astrocytes export glutathione into

the extracellular space where it is hydrolyzed to cysteine and taken

up by neurons for glutathione synthesis. Abbreviations: GGT: gamma

glutamyl synthetase; BBB: blood–brain barrier.

A second unusual aspect of glutathione synthesis in the brain is the

unique metabolic interaction between astrocytes and neurons regarding

uptake of cysteine, the rate-limiting amino acid for glutathione

synthesis (Dringen and Hirrlinger, 2003 and Kranich et al., 1996).

Astrocytes and neurons have different affinities for the uptake of

oxidized and reduced forms of cysteine for glutathione synthesis

(Dringen et al., 2000b). Neurons are unable to take up cystine

(oxidized plasma form of cysteine) but can readily transport reduced

cysteine for glutathione synthesis. In contrast, oxidized cystine is

readily taken up by astrocytes and converted to glutathione as

diagrammed in Fig. 2 (Kranich et al., 1998 and Wang and Cynader,

2000). Because cysteine is the rate-limiting amino acid for

glutathione synthesis, the relative availability of extracellular

cystine and cysteine determines intracellular glutathione

concentrations and resistance to mercury toxicity in astrocytes and

neurons, respectively.

The purpose of the present study was to determine the relative

sensitivity of astrocytes and neurons to Thimerosal (ethyl mercury)

cytotoxicity in vitro and to determine whether Thimerosal

neurotoxicity was associated with depletion of glutathione in

cultured human cells as previously reported for methylmercury

(Sanfeliu et al., 2001). Acute high dose exposures to Thimerosal

( & #956;mol/L) in cultured cells were used to study mechanistic aspects of

Thimerosal toxicity and not intended to mimic exposures of developing

brain cells in vivo to Thimerosal in vaccines (nmol/kg).

MATERIALS AND METHODS

Materials

Culture flasks, 96-well plates, and pipettes were obtained from

Falcon (lin Lakes, NJ, USA). F-12K and MEM culture media were

purchased from ATCC (Manassas, VA, USA) and the RPMI 1640 culture

media, streptomycin, and Dulbecco's Phosphate Buffered Saline were

purchased from Gibco (Grand Island, NY, USA). The TACS™ MTT kit for

cell viability assay was purchased from R & D systems (Minneapolis,

MN, USA). Fetal bovine serum was purchased from HyClone (Logan, UT,

USA). Thimerosal USP, glutathione ethyl ester, cysteine, cystine, N-

acetyl-L-cysteine, L-methionine, and EDTA-trypsin were obtained from

Sigma-Aldrich (St. Louis, MO, USA).

Cell Culture

Human neuroblastoma SH-SY5Y CRL 2266 cells and glioblastoma CRL 2020

cells were purchased from American Type Culture Collection (ATCC,

Manassas, VA) and cultured in T-25 ml flasks in a humidified

incubator at 37 °C with 5% CO2. The neuroblastoma cells were cultured

in 50:50 F-12K media and MEM media containing 15% fetal calf serum

and 1% penicillin/streptomycin. The glioblastoma cells were cultured

in RPMI 1640 media with supplements recommended for this cell line by

ATCC plus 15% fetal calf serum with 1% penicillin/streptomycin.

Cell Viability Assays

Cell viability before and after Thimerosal exposure was assessed

using the MTT assay. Metabolically active mitochondrial

dehydrogenases convert the tetrazolium salt, MTT, to insoluble purple

formazan crystals at a rate that is proportional to cell viability.

For the viability study with Thimerosal exposure, the cultured

neuroblastoma and glioblastoma cells were harvested with 0.25%

trypsin and replated in 96-well microtitre plates at a concentration

of 6.5 × 105 cells/ml and 5 × 105 cells/ml, respectively, in a 100 & #956;l

volume. After a 3-day culture period to reach confluency, 10 & #956;l of

Thimerosal in PBS was added to achieve final concentrations of 2.5,

5, 10 or 20 & #956;mol/L in triplicate wells. Pilot studies conducted with

increasing duration of exposure at 37 °C indicated that a 48 h

incubation period was the threshold for toxicity in the glioblastoma

cells whereas only a 3 h incubation was required to achieve similar

cytotoxicity in the neuroblastoma cells (data not shown). At the end

of the respective incubation periods, 10 & #956;l MTT solution (1:10 v/v)

to each well for 1 h followed by 100 & #956;l of dimethylsulfoxide

detergent solution to lyse the cells and solubilize the formazan

crystals formed in the metabolically active (viable) cells.

Triplicate untreated negative control cells were run together with

the Thimerosal-treated cells. The optical density (OD) was read in

the 96-well plate using the Thermo Max spectrophotometer (Molecular

Devices, Sunnyvale, CA) set at 550 nm (with a reference wavelength of

650 nm). Triplicate wells with reagent only served as background

controls. The results are expressed as the OD after background

subtraction.

Nutrient Supplementation Studies

For the viability studies, cells were exposed to 15 & #956;mol/L Thimerosal

with and without prior incubation with N-acetylcysteine, cystine,

glutathione ethyl ester, or methionine at a final concentration of

100 & #956;mol/L each. N-Acetylcysteine is an acetylated analog of cysteine

that easily crosses the cell membrane and is rapidly deacetylated

inside the cell and utilized for GSH synthesis (Zafarullah et al.,

2003). Glutathione ethyl ester is an esterified form of glutathione

that is able to cross the cell membrane against the concentration

gradient ( et al., 2004). Cystine is the disulfide (oxidized)

form of cysteine that is readily taken up by astrocytes, but not

neurons (Sagara et al., 1993). Since astrocytes are unable to

synthesize GSH from methionine, the addition of methionine to the

media served as a negative control. Triplicate aliquots of

neuroblastoma and astroglial cells were plated in 96-well plates at

concentrations of 8 × 105 cells/ml and 5 × 105 cells/ml,

respectively. The supplements were added to the culture media 45 min

before the addition of Thimerosal and were prepared as 10x

concentrates and added in 10 & #956;L to the cell culture media. Cell

viability after Thimerosal exposure was assessed with the MTT assay

as described above. For HPLC analysis of intracellular glutathione

levels, neuroblastoma and glioblastoma cells were plated in

triplicate in 6-well plates at a density of 6.5 × 105 cells/well and

1 × 106 cells/well, respectively, and cultured for 4 days in order to

generate sufficient cells for HPLC analysis. Triplicate wells were

pooled for analysis.

HPLC Sample Preparation

Briefly, 106 cells were homogenized on ice in 200 & #956;L of phosphate-

buffered saline. To reduce sulfhydryl (thiol) bonds, 50 & #956;l freshly

prepared 1.43 M sodium borohydride solution containing 1.5 & #956;M EDTA,

66 mM NaOH and 10 & #956;l iso-amyl alcohol was added to the homogenate.

After mixing, the solution was incubated in a 40 °C water bath for 30

min with gentle shaking. To precipitate proteins, 250 & #956;L ice cold 10%

meta-phosphoric acid was added, mixed well, and the sample was

incubated for 30 min on ice. After centrifugation at 18,000 × g for

15 min at 4 °C, the supernatant was filtered through a 0.2 & #956;m nylon

membrane filter (PGC Scientific, Frederic, MD). A 20 & #956;l aliquot of

cell extract was directly injected onto the column using Beckman

Autosampler (model 507E).

HPLC with Coulometric Electrochemical Detection

The elution of glutathione was accomplished using HPLC with a

Shimadzu solvent delivery system (ESA model 580) and a reverse phase

C18 column (5 & #956;m; 4.6 mm × 150 mm, MCM Inc., Tokyo, Japan) obtained

from ESA Inc. (Chemsford, MA). A 20 & #956;L aliquot of cell extract was

directly injected onto the column using Beckman autosampler (model

507E). The mobile phase consisted of 50 mM sodium phosphate

monobasic, monohydrate, 1.0 mM ion-pairing reagent octane sulfonic

acid, 2% (v/v) acetonitrile adjusted to pH 2.7 with 85% phosphoric

acid, with isocratic elution at ambient temperature at a flow rate of

1.0 ml/min and a pressure of 120–140 kgf/cm2 (1800–2100 psi). To

assure standardization between sample runs, calibration standards and

reference plasma samples were interspersed at intervals during each

run. The metabolites were quantified using a model 5200A Coulochem II

electrochemical detector (ESA Inc.) equipped with a dual analytical

cell (model 5010) and a guard cell (model 5020). The concentration of

intracellular glutathione was calculated from peak areas and standard

calibration curves using HPLC software and expressed per mg protein.

An aliquot of initial homogenate was used for protein determination

using the BCA protein assay (Pierce Inc., Rockford, IL).

Statistics

Values are expressed as the mean ± standard deviation. One way ANOVA

was used to determine significant differences between groups and the

Bonferroni t-test for pairwise comparisons using Sigma Stat 2.0

software. Treatment related differences were considered to be

significant at p < 0.05.

RESULTS

Dose–Response Characteristics of Thimerosal in Glioblastoma and

Neuroblastoma Cells

Fig. 3A and B shows the dose response characteristics of triplicate

cultures of the glioblastoma and neuroblastoma cells, respectively.

In both cell lines, a progressive increase in cytotoxicity (decrease

in viability) was observed when Thimerosal dose was progressively

doubled from 2.5 & #956;mol/L to 5, 10 and 20 & #956;mol/L. Viability was reduced

more than 50% in both cell lines with exposure to 10 & #956;mol/L

Thimerosal and less than 10% of cells survived a dose of 20 & #956;mol/L.

Although the shape of the dose response is similar between the two

cell types, the dose response curve for the neuroblastoma cells

occurred with a only a 3 h exposure whereas the glioblastoma cell

required 48 h to exhibit similar cytotoxic effects at the same dose.

These results demonstrate that the neuroblastoma cells are much more

sensitive to Thimerosal cytoxicity than are glioblastoma cells.

(27K)

Fig. 3. Viability of glioblastoma cells (A) and neuroblastoma cells

( with increasing concentrations of Thimerosal in the media.

Asterisks indicate significant differences from control cells without

Thimerosal treatment (n = 3, p < 0.01).

Neuroprotective Effect of N-Acetylcysteine, Cystine, and Glutathione

Ethyl Ester

Based on the viability assays, 15 & #956;mol/L Thimerosal was selected as

the dose for the supplementation studies since a significant level of

toxicity was observed in both cell lines.

Glioblastoma Cells

Triplicate cultures were pretreated for 1 h with 100 & #956;mol/L N-

acetylcysteine (NAC), glutathione ethyl ester, cystine, or methionine

before addition of 15 & #956;mol/L Thimerosal to the culture medium. As

shown in Fig. 4A, 15 & #956;mol/L Thimerosal alone induced approximately 3-

fold decrease in cell viability whereas pretreatment with either

cystine, glutathione, or NAC provided significant protection from

cell death. The failure of methionine to provide precursors for GSH

synthesis is consistent with the lack of cystathionine & #947; lyase

activity in these cells.

(44K)

Fig. 4. Viability of glioblastoma cells (A) and neuroblastoma cells

( without (control) and with exposure to 15 & #956;mol/L Thimerosal alone

for 48 h or Thimerosal treatment after a 45 min pretreatment with 100

& #956;mol/L of N-acetyl cysteine (NAC), glutathione ethyl ester (GSH)

cystine, or methionine. Asterisks indicate significant differences

between cells treated with Thimerosal alone and cells pretreated with

indicated nutrients (n = 3, p < 0.05).

Neuroblastoma Cells

Triplicate cultures of neuroblastoma cells were pretreated for 1 h

with the same supplements as the glioblastoma cells. Fig. 4B

demonstrates that Thimerosal alone induced more than a 6-fold

decrease in viability, confirming the increased vulnerability of

neuroblastoma cells to Thimerosal relative to the glioblastoma cells.

Both NAC and glutathione ethyl ester provided significant protection

against cell death whereas cystine and methionine were without

effect. The lack of effect of cystine is consistent with previous

reports that neurons can take up cysteine, but not cystine (Sagara et

al., 1993). As expected, methionine afforded no protection.

Glutathione Depletion with Thimerosal Exposure: Preservation of

Intracellular GSH with Nutritional Supplementation

The baseline level of intracellular glutathione was 39 & #956;mol/L in the

glioblastoma cells compared to only 26 & #956;mol/L in the neuroblastoma

cells (Fig. 5A). Exposure to15 & #956;mol/L Thimerosal for 1 h caused less

than a 50% decrease in intracellular glutathione levels in the

glioblastoma cells whereas the same exposure induced more than an 8-

fold-decrease in the neuroblastoma cells. Pretreatment with NAC or

glutathione ethyl ester completely prevented the Thimerosal-induced

depletion in glutathione in neuroblastoma cells (Fig. 5B). In the

glioblastoma cells, pretreatment with NAC completely prevented the

Thimerosal-induced glutathione depletion and glutathione ethyl ester

was partially protective.

(40K)

Fig. 5. Concentration of intracellular glutathione in 106

glioblastoma cells (A) and neuroblastoma cells ( without (control)

and with 15 & #956;mol/L Thimerosal alone or Thimerosal after pretreatment

with 100 & #956;mol/L of N-acetyl cysteine (NAC), glutathione ethyl ester

(GSH) in a representative experiment that was repeated with similar

results.

DISCUSSION

Considerable concern has been expressed recently over the cumulative

dose of mercury given to children through routine immunizations given

in the 1990s. The source of mercury in vaccines is the antimicrobial

preservative, Thimerosal, containing 49.9% ethyl mercury by weight.

All forms of mercury are well known to be neurotoxic, especially

during early brain development (Costa et al., 2004). The high

affinity binding of mercuric compounds to the thiol (SH) group of

cysteines in essential proteins is thought to be the basis for

mercury-induced cytotoxicity. In vivo studies in rodents have shown

that ethyl mercury is able to cross the cell membrane and then is

converted intracellularly to inorganic mercury which accumulates

preferentially in the brain and kidney (Magos et al., 1985).

Intracellular accumulation of inorganic mercury was shown to be

higher for ethyl compared to methylmercury with equimolar exposure,

although the clearance rate of ethylmercury was faster than

methylmercury (Magos et al., 1985). A recent in vitro study of

Thimerosal exposure to fibroblasts and human cerebral cortical neuron

cell lines demonstrated that short term exposure in concentrations

similar to those used in the present study induced DNA strand breaks,

membrane damage, caspase-3 activation, and cell death (Baskin et al.,

2003). The purpose of the present study was to determine whether the

mechanism of ethylmercury toxicity was similar to that previously

reported for methylmercury. Human glioblastoma and neuroblastoma cell

lines were used as surrogates for astrocytes and neurons,

respectively, based on a previous study demonstrating similar dose–

response profiles to methyl mercury between primary cultures and

transformed cell lines (Sanfeliu et al., 2001).

Glutathione provides the major intracellular defense against ROS and

oxidative stress-induced cell damage and apoptosis (Meister, 1995).

Agents or conditions that deplete mitochondrial glutathione will

indirectly increase ROS levels and induce cell death in a variety of

cell types ( et al., 2001 and Marchetti et al., 1997). Mercury

and other heavy metals are well known to increase oxidative stress

and deplete intracellular glutathione (Naganuma et al., 1990). A

major unanswered question is whether mercury-induced depletion of

glutathione precedes the increase in ROS or whether mercury-induced

ROS induces glutathione depletion. In thymocytes, mitochondrial

glutathione depletion was shown to precede the increase in ROS

associated with loss of viability and apoptosis (Macho et al., 1997).

Whether mercury-induced depletion of glutathione is the initiating

factor for increased oxidative stress and cell death in brain cells

has not yet been evaluated.

A recent in vitro study of Thimerosal immunotoxicity using

immortalized Jurkat T cells demonstrated an increase in reactive

oxygen species and a decrease in intracellular glutathione with

increasing concentrations of Thimerosal (Makani et al., 2002).

Thimerosal, but not thiosalacylic acid (the non-mercury component of

Thimerosal), induced apoptotic cell death in T cells in a

concentration-dependent manner as evidenced by mitochondrial release

of cytochrome c, apoptosis activating factor, and activation of

caspases 9 and 3. Exogenous glutathione inhibited activation of these

caspases and prevented cell death. These results suggest that, at

least in T cells, Thimerosal induces oxidative stress and apoptosis

by activating mitochondrial cell death pathways. A subsequent study

using cultured human neuron and fibroblast cell lines similarly

showed that low micromolar concentrations of Thimerosal induced DNA

strand breaks, caspase-3 activation, membrane damage and cell death

(Baskin et al., 2003).

In the present study, we evaluated glioblastoma cells and

neuroblastoma cells in culture to determine the relative sensitivity

of each cell type to Thimerosal-induced oxidative stress and cell

death. At equimolar concentrations of Thimerosal, the neurons were

found to be much more sensitive to Thimerosal-induced cell death than

the astrocytes. In the neuronal cell line, viability was

significantly reduced in a concentration-dependent manner at 2.5, 5,

10, and 20 & #956;mol/L Thimerosal after only a 3 h exposure, whereas the

astrocytes required a full 48 h exposure for a similar loss of

viability (Fig. 3). These results duplicate observations in the same

cell lines exposed to similar concentrations of methyl mercury and

suggest that the mechanism of ethyl- and methylmercury neurotoxicity

is similar. The addition of either N-acetylcysteine or gluthatione

ethyl ester (100 & #956;mol/L) to the culture medium 1 h before adding 15

& #956;mol/L Thimerosal conferred significant protection against

cytotoxicity in both cell lines (Fig. 4). It is likely that the

extracellular NAC and glutathione provided partial protection by

complexing with the Thimerosal in the culture medium as well as by

increasing intracellular glutathione levels. The oxidized form of

cysteine (cystine) was protective in astrocytes, but not neurons,

consistent with facilitated membrane transport of cystine in

astrocytes (Kranich et al., 1998). Neurons depend on glutathione

synthesized in the astrocytes and released extracellularly where it

is hydrolyzed to cysteinylglycine and cysteine by ectoenzymes to

provide neurons with necessary precursors for intracellular

gluthathione synthesis (Dringen et al., 1999). In both cell lines,

methionine provided no protection against Thimerosal toxicity

confirming the inability of either cell type to synthesize cysteine

(and glutathione) from methionine.

The intracellular concentration of glutathione before supplementation

was 30% lower in neuroblastoma cells compared to the glioblastoma

cells. The lower baseline glutathione concentration in the neuronal

cell line was associated with increased sensitivity to Thimerosal

cytotoxicity (Fig. 5). Thus, sensitivity to Thimerosal was directly

proportional to the basal intracellular glutathione concentration. In

co-culture studies, astrocytes have been shown to protect neurons

against the toxicity of oxidative stress (Dringen et al., 2000a). The

provision of glutathione precursors to neurons is a possible

explanation for the protective effect of astrocytes. Recent results

have confirmed the primary role of astrocytes in glutathione

metabolism and antioxidant defense in the brain (Dringen, 2000).

Depletion of astrocyte glutathione would therefore indirectly induce

oxidative cell death in neurons by depletion of essential glutathione

precursors.

In summary, we have shown that human glioblastoma cells are more

resistant to Thimerosal cytotoxicity than neuroblastoma cells at

doses in the low micromolar range and that the resistance is

correlated with higher intracellular levels of intracellular

glutathione. The significant protection by NAC and glutathione ethyl

ester against Thimerosal cytotoxicity suggests the possibility that

supplementation with glutathione precursors might be protective

against mercury exposures in vivo. Numerous clinical studies have

demonstrated the efficacy of NAC in increasing intracellular

glutathione levels and reducing oxidative stress in humans (

and Luo, 1998 and Badaloo et al., 2002). Since cytotoxicity with both

ethyl- and methylmercury have been shown to be mediated by

glutathione depletion, dietary supplements that increase

intracellular glutathione could be envisioned as an effective

intervention to reduce previous or anticipated exposure to mercury.

This approach would be especially valuable in the elderly and in

pregnant women before receiving flu vaccinations, in pregnant women

receiving Rho D immunoglobulin shots, and individuals who regularly

consume mercury-containing fish.

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H. Kodama, Changes in cystathionine gamma-lyase in various regions of

rat brain during development, Biochem Mol Biol Int 35 (1995) (6), pp.

1331–1338.

Badaloo et al., 2002 A. Badaloo, M. Reid, T. Forrester, W.C. Heird

and F. Jahoor, Cysteine supplementation improves the erythrocyte

glutathione synthesis rate in children with severe edematous

malnutrition, Am J Clin Nutr 76 (2002) (3), pp. 646–652.

Ball et al., 2001 L.K. Ball, R. Ball and R.D. Pratt, An assessment of

thimerosal use in childhood vaccines, Pediatrics 107 (2001) (5), pp.

1147–1154.

Baskin et al., 2003 D.S. Baskin, H. Ngo and V.V. Didenko, Thimerosal

induces DNA breaks, caspase-3 activation, membrane damage, and cell

death in cultured human neurons and fibroblasts, Toxicol Sci 74

(2003) (2), pp. 361–368.

Costa et al., 2004 L.G. Costa, M. Aschner, A. Vitalone, T. Syversen

and O.P. Soldin, Developmental neuropathology of environmental

agents, Annu Rev Pharmacol Toxicol 44 (2004), pp. 87–110.

Dringen, 2000 R. Dringen, Metabolism and functions of glutathione in

brain, Progr Neurobiol 62 (2000) (6), pp. 649–671. Abstract | Full

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Dringen et al., 2000a R. Dringen, J.M. Gutterer and J. Hirrlinger,

Glutathione metabolism in brain metabolic interaction between

astrocytes and neurons in the defense against reactive oxygen

species, Eur J Biochem 267 (2000) (16), pp. 4912–4916.

Dringen et al., 2000b R. Dringen, J.M. Gutterer and J. Hirrlinger,

Glutathione metabolism in brain metabolic interaction between

astrocytes and neurons in the defense against reactive oxygen

species, Eur J Biochem 267 (2000) (16), pp. 4912–4916.

Dringen and Hirrlinger, 2003 R. Dringen and J. Hirrlinger,

Glutathione pathways in the brain, Biol Chem 384 (2003) (4), pp. 505–

516.

Dringen et al., 1999 R. Dringen, B. Pfeiffer and B. Hamprecht,

Synthesis of the antioxidant glutathione in neurons: supply by

astrocytes of CysGly as precursor for neuronal glutathione, J

Neurosci 19 (1999) (2), pp. 562–569.

Kranich et al., 1998 O. Kranich, R. Dringen, M. Sandberg and B.

Hamprecht, Utilization of cysteine and cysteine precursors for the

synthesis of glutathione in astroglial cultures: preference for

cystine, Glia 22 (1998) (1), pp. 11–18.

Kranich et al., 1996 O. Kranich, B. Hamprecht and R. Dringen,

Different preferences in the utilization of amino acids for

glutathione synthesis in cultured neurons and astroglial cells

derived from rat brain, Neurosci Lett 219 (1996) (3), pp. 211–214.

Abstract | Full Text + Links | PDF (81 K)

Lu, 1998 S.C. Lu, Regulation of hepatic glutathione synthesis, Semin

Liver Dis 18 (1998) (4), pp. 331–343.

Macho et al., 1997 A. Macho, T. Hirsch, I. Marzo, P. Marchetti, B.

Dallaporta and S.A. Susin et al., Glutathione depletion is an early

and calcium elevation is a late event of thymocyte apoptosis, J

Immunol 158 (1997) (10), pp. 4612–4619.

Magos et al., 1985 L. Magos, A.W. Brown, S. Sparrow, E. , R.T.

Snowden and W.R. Skipp, The comparative toxicology of ethyl- and

methylmercury, Arch Toxicol 57 (1985) (4), pp. 260–267.

Makani et al., 2002 S. Makani, S. Gollapudi, L. Yel, S. Chiplunkar

and S. Gupta, Biochemical and molecular basis of thimerosal-induced

apoptosis in T cells: a major role of mitochondrial pathway, Genes

Immun 3 (2002) (5), pp. 270–278.

Marchetti et al., 1997 P. Marchetti, D. Decaudin, A. Macho, N.

Zamzami, T. Hirsch and S.A. Susin et al., Redox regulation of

apoptosis: impact of thiol oxidation status on mitochondrial

function, Eur J Immunol 27 (1997) (1), pp. 289–296.

Meister, 1995 A. Meister, Glutathione metabolism, Methods Enzymol 251

(1995), pp. 3–7.

Naganuma et al., 1990 A. Naganuma, M.E. and A. Meister,

Cellular glutathione as a determinant of sensitivity to mercuric

chloride toxicity. Prevention of toxicity by giving glutathione

monoester, Biochem Pharmacol 40 (1990) (4), pp. 693–697.

Public Health Service, 1999 Public Health Service, Department of

Health and Human Services & American Academy of Pediatrics. Morb

Mortal Weekly Rep 1999;48:563–4.

Sagara et al., 1993 J.I. Sagara, K. Miura and S. Bannai, Maintenance

of neuronal glutathione by glial cells, J Neurochem 61 (1993) (5),

pp. 1672–1676.

Sanfeliu et al., 2003 C. Sanfeliu, J. Sebastia, R. Cristofol and E.

-Farre, Neurotoxicity of organomercurial compounds, Neurotox

Res 5 (2003) (4), pp. 283–305.

Sanfeliu et al., 2001 C. Sanfeliu, J. Sebastia and S.U. Ki,

Methylmercury neurotoxicity in cultures of human neurons, astrocytes,

neuroblastoma cells, Neurotoxicology 22 (2001) (3), pp. 317–327.

Abstract | Full Text + Links | PDF (624 K)

Wang and Cynader, 2000 X.F. Wang and M.S. Cynader, Astrocytes provide

cysteine to neurons by releasing glutathione, J Neurochem 74 (2000)

(4), pp. 1434–1442.

Zafarullah et al., 2003 M. Zafarullah, W.Q. Li, J. Sylvester and M.

Ahmad, Molecular mechanisms of N-acetylcysteine actions, Cell Mol

Life Sci 60 (2003) (1), pp. 6–20.

Corresponding author. Tel.: +1 501 364 4665; fax: +1 501 364 5107.

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>

>

> Theresa:

>

> I agree with you, the EWG report seems remarkable. How we can find

> out more about the protocol that Dr. followed.

>

> It would be completely amazing, not short of a miracle if our

> children could be cured of apraxia...

>

> Does anyone in our group have more information regarding this EWG

> report and the protocol? I'm up for discussion on this important

> issue.

>

> G.-- Do you have any insight regarding the EWG Report and the

> protocol? What are your thoughts?

>

> Thanks,

>

>

>

>

> ,

[Guest guest]

WOW!!

Thank you,

theresa looking for an interperter(SP?)

I am taking this along with Colleen's great post to my ped

[ ] Re:EWG Report ---- Autism report news release

12.13.04

To All:

This is the journal article as it appeared in the Journal of

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doi:10.1016/j.neuro.2004.07.012

Copyright © 2004 Elsevier Inc. All rights reserved.

Thimerosal Neurotoxicity is Associated with Glutathione Depletion:

Protection with Glutathione Precursors

S.J. 1, , , Slikker III2, Stepan Melnyk1,

New2, Marta Pogribna2 and Stefanie Jernigan1

1Department of Pediatrics, University of Arkansas for Medical

Sciences and Arkansas Children's Hospital Research Institute, Little

Rock, AR 72202, USA

2Division of Biochemical Toxicology, National Center for

Toxicological Research, Jefferson, AR 72079, USA

Received 24 May 2004; accepted 28 July 2004. Available online 29

September 2004.

Abstract

Thimerosol is an antiseptic containing 49.5% ethyl mercury that has

been used for years as a preservative in many infant vaccines and in

flu vaccines. Environmental methyl mercury has been shown to be

highly neurotoxic, especially to the developing brain. Because

mercury has a high affinity for thiol (sulfhydryl (SH)) groups, the

thiol-containing antioxidant, glutathione (GSH), provides the major

intracellular defense against mercury-induced neurotoxicity. Cultured

neuroblastoma cells were found to have lower levels of GSH and

increased sensitivity to thimerosol toxicity compared to glioblastoma

cells that have higher basal levels of intracellular GSH. Thimerosal-

induced cytotoxicity was associated with depletion of intracellular

GSH in both cell lines. Pretreatment with 100 & #956;M glutathione ethyl

ester or N-acetylcysteine (NAC), but not methionine, resulted in a

significant increase in intracellular GSH in both cell types.

Further, pretreatment of the cells with glutathione ethyl ester or

NAC prevented cytotoxicity with exposure to 15 & #956;M Thimerosal.

Although Thimerosal has been recently removed from most children's

vaccines, it is still present in flu vaccines given to pregnant

women, the elderly, and to children in developing countries. The

potential protective effect of GSH or NAC against mercury toxicity

warrants further research as possible adjunct therapy to individuals

still receiving Thimerosal-containing vaccinations.

Keywords: Thimerosal; Neurotoxicity; Glutathione; N-acetylcysteine

Article Outline

INTRODUCTION

MATERIALS AND METHODS

Materials

Cell Culture

Cell Viability Assays

Nutrient Supplementation Studies

HPLC Sample Preparation

HPLC with Coulometric Electrochemical Detection

Statistics

RESULTS

Dose-Response Characteristics of Thimerosal in Glioblastoma and

Neuroblastoma Cells

Neuroprotective Effect of N-Acetylcysteine, Cystine, and Glutathione

Ethyl Ester

Glioblastoma Cells

Neuroblastoma Cells

Glutathione Depletion with Thimerosal Exposure: Preservation of

Intracellular GSH with Nutritional Supplementation

DISCUSSION

References

INTRODUCTION

Thimerosal (sodium ethylmercurithiosalicylate) was developed by Eli

Lilly in the 1930s as a effective bacteriostatic and fungistatic

preservative and has been widely used in multidose vials of vaccines

and in ophthalmic, otic, nasal, and topical products. Until the

removal of Thimerosal from most pediatric vaccines in 2001, the

largest human exposure in the US ( & #956;g/kg body weight) was in children

under 18 months of age undergoing routine childhood immunization

schedules. Prior to 2001, a child may have received a cumulative dose

of over 200 & #956;g/kg in the first 18 months of life (Ball et al., 2001).

Although the neurotoxicity of methyl mercury has been relatively well

studied, limited information is available on the relative

neurodevelopmental toxicity of ethylmercury, the mercury metabolite

of Thimerosal. Based on the known toxicity of methylmercury, the

cumulative ethylmercury exposure to US pediatric populations in

Thimerosal-containing vaccinations was re-examined in 1999 and found

to exceed EPA recommended guidelines (Ball et al., 2001). Following

recommendations by the American Academy of Pediatrics and the US

Public Health Service (Public Health Service, 1999), Thimerosal was

subsequently removed as a preservative from most children's vaccines

in the US. However, influenza vaccines and Rho D immunoglobulin shots

containing Thimerosal are still recommended to pregnant women, and

many vaccines given to children in developing countries still contain

Thimerosal. The present study was undertaken to better understand the

mechanisms underlying Thimerosal toxicity to neurons and astrocytes,

the primary CNS targets for organic mercury (Sanfeliu et al., 2001).

A better understanding of the neurotoxic mechanism is a necessary

prerequisite for the design of intervention strategies for prevention

and for the identification of genetic variants that could increase

sensitivity to Thimerosal.

Previous mechanistic studies of methylmercury toxicity in astrocytes

and neurons have implicated reactive oxygen species (ROS) and

depletion of intracellular glutathione as major contributors to

mercury-induced cytotoxicity (Sanfeliu et al., 2001). Organic mercury

has a high affinity for the thiol (SH) group on glutathione, a

tripeptide composed of cysteine, glutamate, and glycine (Sanfeliu et

al., 2003). The cysteine moiety of glutathione carries the active

thiol group that binds and detoxifies a variety of heavy metals,

including organic and inorganic mercury. Normally, the intracellular

concentration of glutathione is extremely high, in the mM range

(Meister, 1995); however, with depletion of this essential

antioxidant, excess free mercury is available to bind to cysteine

thiol groups present in essential cellular proteins, leading to

functional inactivation and cytotoxicity.

The synthesis of glutathione in the brain is unique in that brain

cells do not express cystathionine gamma lyase, an enzyme in the

transsulfuration pathway involved in glutathione synthesis (Awata et

al., 1995). As a result, brain cells cannot synthesize cysteine, the

rate limiting amino acid for glutathione synthesis. Thus, the brain

is dependent on the liver to synthesize and export cysteine for

uptake and utilization by astrocytes and neurons for adequate

glutathione synthesis (Lu, 1998). In contrast to the brain, the liver

expresses the complete transsulfuration pathway from methionine to

cysteine and glutathione as diagrammed in Fig. 1. Glutathione

synthesized in the liver is exported to the plasma where it is

immediately degraded to cysteinylglycine and cysteine (Lu, 1998).

Cysteine is converted to cystine in the oxidizing environment of the

plasma and subsequently transported to the brain for intracellular

glutathione synthesis (Fig. 2).

(29K)

Fig. 1. Pathways of glutathione synthesis. The liver expresses the

complete pathway from methionine through homocysteine and cysteine to

glutathione. Astrocytes and neurons do not express the enzyme

cystathionine lyase and therefore are unable to synthesize cysteine.

As a result, astrocytes and neurons are dependent on plasma cysteine

derived primarily from the liver to synthesize glutathione.

(6K)

Fig. 2. Interaction between astrocytes and neurons for glutathione

synthesis. Glutathione exported from the liver is hydrolyzed to

cysteinylglycine and cysteine. In the plasma, cysteine is oxidized to

cystine and transported across the BBB and taken up by astrocytes and

used to synthesize glutathione. Astrocytes export glutathione into

the extracellular space where it is hydrolyzed to cysteine and taken

up by neurons for glutathione synthesis. Abbreviations: GGT: gamma

glutamyl synthetase; BBB: blood-brain barrier.

A second unusual aspect of glutathione synthesis in the brain is the

unique metabolic interaction between astrocytes and neurons regarding

uptake of cysteine, the rate-limiting amino acid for glutathione

synthesis (Dringen and Hirrlinger, 2003 and Kranich et al., 1996).

Astrocytes and neurons have different affinities for the uptake of

oxidized and reduced forms of cysteine for glutathione synthesis

(Dringen et al., 2000b). Neurons are unable to take up cystine

(oxidized plasma form of cysteine) but can readily transport reduced

cysteine for glutathione synthesis. In contrast, oxidized cystine is

readily taken up by astrocytes and converted to glutathione as

diagrammed in Fig. 2 (Kranich et al., 1998 and Wang and Cynader,

2000). Because cysteine is the rate-limiting amino acid for

glutathione synthesis, the relative availability of extracellular

cystine and cysteine determines intracellular glutathione

concentrations and resistance to mercury toxicity in astrocytes and

neurons, respectively.

The purpose of the present study was to determine the relative

sensitivity of astrocytes and neurons to Thimerosal (ethyl mercury)

cytotoxicity in vitro and to determine whether Thimerosal

neurotoxicity was associated with depletion of glutathione in

cultured human cells as previously reported for methylmercury

(Sanfeliu et al., 2001). Acute high dose exposures to Thimerosal

( & #956;mol/L) in cultured cells were used to study mechanistic aspects of

Thimerosal toxicity and not intended to mimic exposures of developing

brain cells in vivo to Thimerosal in vaccines (nmol/kg).

MATERIALS AND METHODS

Materials

Culture flasks, 96-well plates, and pipettes were obtained from

Falcon (lin Lakes, NJ, USA). F-12K and MEM culture media were

purchased from ATCC (Manassas, VA, USA) and the RPMI 1640 culture

media, streptomycin, and Dulbecco's Phosphate Buffered Saline were

purchased from Gibco (Grand Island, NY, USA). The TACST MTT kit for

cell viability assay was purchased from R & D systems (Minneapolis,

MN, USA). Fetal bovine serum was purchased from HyClone (Logan, UT,

USA). Thimerosal USP, glutathione ethyl ester, cysteine, cystine, N-

acetyl-L-cysteine, L-methionine, and EDTA-trypsin were obtained from

Sigma-Aldrich (St. Louis, MO, USA).

Cell Culture

Human neuroblastoma SH-SY5Y CRL 2266 cells and glioblastoma CRL 2020

cells were purchased from American Type Culture Collection (ATCC,

Manassas, VA) and cultured in T-25 ml flasks in a humidified

incubator at 37 °C with 5% CO2. The neuroblastoma cells were cultured

in 50:50 F-12K media and MEM media containing 15% fetal calf serum

and 1% penicillin/streptomycin. The glioblastoma cells were cultured

in RPMI 1640 media with supplements recommended for this cell line by

ATCC plus 15% fetal calf serum with 1% penicillin/streptomycin.

Cell Viability Assays

Cell viability before and after Thimerosal exposure was assessed

using the MTT assay. Metabolically active mitochondrial

dehydrogenases convert the tetrazolium salt, MTT, to insoluble purple

formazan crystals at a rate that is proportional to cell viability.

For the viability study with Thimerosal exposure, the cultured

neuroblastoma and glioblastoma cells were harvested with 0.25%

trypsin and replated in 96-well microtitre plates at a concentration

of 6.5 × 105 cells/ml and 5 × 105 cells/ml, respectively, in a 100 & #956;l

volume. After a 3-day culture period to reach confluency, 10 & #956;l of

Thimerosal in PBS was added to achieve final concentrations of 2.5,

5, 10 or 20 & #956;mol/L in triplicate wells. Pilot studies conducted with

increasing duration of exposure at 37 °C indicated that a 48 h

incubation period was the threshold for toxicity in the glioblastoma

cells whereas only a 3 h incubation was required to achieve similar

cytotoxicity in the neuroblastoma cells (data not shown). At the end

of the respective incubation periods, 10 & #956;l MTT solution (1:10 v/v)

to each well for 1 h followed by 100 & #956;l of dimethylsulfoxide

detergent solution to lyse the cells and solubilize the formazan

crystals formed in the metabolically active (viable) cells.

Triplicate untreated negative control cells were run together with

the Thimerosal-treated cells. The optical density (OD) was read in

the 96-well plate using the Thermo Max spectrophotometer (Molecular

Devices, Sunnyvale, CA) set at 550 nm (with a reference wavelength of

650 nm). Triplicate wells with reagent only served as background

controls. The results are expressed as the OD after background

subtraction.

Nutrient Supplementation Studies

For the viability studies, cells were exposed to 15 & #956;mol/L Thimerosal

with and without prior incubation with N-acetylcysteine, cystine,

glutathione ethyl ester, or methionine at a final concentration of

100 & #956;mol/L each. N-Acetylcysteine is an acetylated analog of cysteine

that easily crosses the cell membrane and is rapidly deacetylated

inside the cell and utilized for GSH synthesis (Zafarullah et al.,

2003). Glutathione ethyl ester is an esterified form of glutathione

that is able to cross the cell membrane against the concentration

gradient ( et al., 2004). Cystine is the disulfide (oxidized)

form of cysteine that is readily taken up by astrocytes, but not

neurons (Sagara et al., 1993). Since astrocytes are unable to

synthesize GSH from methionine, the addition of methionine to the

media served as a negative control. Triplicate aliquots of

neuroblastoma and astroglial cells were plated in 96-well plates at

concentrations of 8 × 105 cells/ml and 5 × 105 cells/ml,

respectively. The supplements were added to the culture media 45 min

before the addition of Thimerosal and were prepared as 10x

concentrates and added in 10 & #956;L to the cell culture media. Cell

viability after Thimerosal exposure was assessed with the MTT assay

as described above. For HPLC analysis of intracellular glutathione

levels, neuroblastoma and glioblastoma cells were plated in

triplicate in 6-well plates at a density of 6.5 × 105 cells/well and

1 × 106 cells/well, respectively, and cultured for 4 days in order to

generate sufficient cells for HPLC analysis. Triplicate wells were

pooled for analysis.

HPLC Sample Preparation

Briefly, 106 cells were homogenized on ice in 200 & #956;L of phosphate-

buffered saline. To reduce sulfhydryl (thiol) bonds, 50 & #956;l freshly

prepared 1.43 M sodium borohydride solution containing 1.5 & #956;M EDTA,

66 mM NaOH and 10 & #956;l iso-amyl alcohol was added to the homogenate.

After mixing, the solution was incubated in a 40 °C water bath for 30

min with gentle shaking. To precipitate proteins, 250 & #956;L ice cold 10%

meta-phosphoric acid was added, mixed well, and the sample was

incubated for 30 min on ice. After centrifugation at 18,000 × g for

15 min at 4 °C, the supernatant was filtered through a 0.2 & #956;m nylon

membrane filter (PGC Scientific, Frederic, MD). A 20 & #956;l aliquot of

cell extract was directly injected onto the column using Beckman

Autosampler (model 507E).

HPLC with Coulometric Electrochemical Detection

The elution of glutathione was accomplished using HPLC with a

Shimadzu solvent delivery system (ESA model 580) and a reverse phase

C18 column (5 & #956;m; 4.6 mm × 150 mm, MCM Inc., Tokyo, Japan) obtained

from ESA Inc. (Chemsford, MA). A 20 & #956;L aliquot of cell extract was

directly injected onto the column using Beckman autosampler (model

507E). The mobile phase consisted of 50 mM sodium phosphate

monobasic, monohydrate, 1.0 mM ion-pairing reagent octane sulfonic

acid, 2% (v/v) acetonitrile adjusted to pH 2.7 with 85% phosphoric

acid, with isocratic elution at ambient temperature at a flow rate of

1.0 ml/min and a pressure of 120-140 kgf/cm2 (1800-2100 psi). To

assure standardization between sample runs, calibration standards and

reference plasma samples were interspersed at intervals during each

run. The metabolites were quantified using a model 5200A Coulochem II

electrochemical detector (ESA Inc.) equipped with a dual analytical

cell (model 5010) and a guard cell (model 5020). The concentration of

intracellular glutathione was calculated from peak areas and standard

calibration curves using HPLC software and expressed per mg protein.

An aliquot of initial homogenate was used for protein determination

using the BCA protein assay (Pierce Inc., Rockford, IL).

Statistics

Values are expressed as the mean ± standard deviation. One way ANOVA

was used to determine significant differences between groups and the

Bonferroni t-test for pairwise comparisons using Sigma Stat 2.0

software. Treatment related differences were considered to be

significant at p < 0.05.

RESULTS

Dose-Response Characteristics of Thimerosal in Glioblastoma and

Neuroblastoma Cells

Fig. 3A and B shows the dose response characteristics of triplicate

cultures of the glioblastoma and neuroblastoma cells, respectively.

In both cell lines, a progressive increase in cytotoxicity (decrease

in viability) was observed when Thimerosal dose was progressively

doubled from 2.5 & #956;mol/L to 5, 10 and 20 & #956;mol/L. Viability was

reduced

more than 50% in both cell lines with exposure to 10 & #956;mol/L

Thimerosal and less than 10% of cells survived a dose of 20 & #956;mol/L.

Although the shape of the dose response is similar between the two

cell types, the dose response curve for the neuroblastoma cells

occurred with a only a 3 h exposure whereas the glioblastoma cell

required 48 h to exhibit similar cytotoxic effects at the same dose.

These results demonstrate that the neuroblastoma cells are much more

sensitive to Thimerosal cytoxicity than are glioblastoma cells.

(27K)

Fig. 3. Viability of glioblastoma cells (A) and neuroblastoma cells

( with increasing concentrations of Thimerosal in the media.

Asterisks indicate significant differences from control cells without

Thimerosal treatment (n = 3, p < 0.01).

Neuroprotective Effect of N-Acetylcysteine, Cystine, and Glutathione

Ethyl Ester

Based on the viability assays, 15 & #956;mol/L Thimerosal was selected as

the dose for the supplementation studies since a significant level of

toxicity was observed in both cell lines.

Glioblastoma Cells

Triplicate cultures were pretreated for 1 h with 100 & #956;mol/L N-

acetylcysteine (NAC), glutathione ethyl ester, cystine, or methionine

before addition of 15 & #956;mol/L Thimerosal to the culture medium. As

shown in Fig. 4A, 15 & #956;mol/L Thimerosal alone induced approximately 3-

fold decrease in cell viability whereas pretreatment with either

cystine, glutathione, or NAC provided significant protection from

cell death. The failure of methionine to provide precursors for GSH

synthesis is consistent with the lack of cystathionine & #947; lyase

activity in these cells.

(44K)

Fig. 4. Viability of glioblastoma cells (A) and neuroblastoma cells

( without (control) and with exposure to 15 & #956;mol/L Thimerosal alone

for 48 h or Thimerosal treatment after a 45 min pretreatment with 100

& #956;mol/L of N-acetyl cysteine (NAC), glutathione ethyl ester (GSH)

cystine, or methionine. Asterisks indicate significant differences

between cells treated with Thimerosal alone and cells pretreated with

indicated nutrients (n = 3, p < 0.05).

Neuroblastoma Cells

Triplicate cultures of neuroblastoma cells were pretreated for 1 h

with the same supplements as the glioblastoma cells. Fig. 4B

demonstrates that Thimerosal alone induced more than a 6-fold

decrease in viability, confirming the increased vulnerability of

neuroblastoma cells to Thimerosal relative to the glioblastoma cells.

Both NAC and glutathione ethyl ester provided significant protection

against cell death whereas cystine and methionine were without

effect. The lack of effect of cystine is consistent with previous

reports that neurons can take up cysteine, but not cystine (Sagara et

al., 1993). As expected, methionine afforded no protection.

Glutathione Depletion with Thimerosal Exposure: Preservation of

Intracellular GSH with Nutritional Supplementation

The baseline level of intracellular glutathione was 39 & #956;mol/L in the

glioblastoma cells compared to only 26 & #956;mol/L in the neuroblastoma

cells (Fig. 5A). Exposure to15 & #956;mol/L Thimerosal for 1 h caused less

than a 50% decrease in intracellular glutathione levels in the

glioblastoma cells whereas the same exposure induced more than an 8-

fold-decrease in the neuroblastoma cells. Pretreatment with NAC or

glutathione ethyl ester completely prevented the Thimerosal-induced

depletion in glutathione in neuroblastoma cells (Fig. 5B). In the

glioblastoma cells, pretreatment with NAC completely prevented the

Thimerosal-induced glutathione depletion and glutathione ethyl ester

was partially protective.

(40K)

Fig. 5. Concentration of intracellular glutathione in 106

glioblastoma cells (A) and neuroblastoma cells ( without (control)

and with 15 & #956;mol/L Thimerosal alone or Thimerosal after pretreatment

with 100 & #956;mol/L of N-acetyl cysteine (NAC), glutathione ethyl ester

(GSH) in a representative experiment that was repeated with similar

results.

DISCUSSION

Considerable concern has been expressed recently over the cumulative

dose of mercury given to children through routine immunizations given

in the 1990s. The source of mercury in vaccines is the antimicrobial

preservative, Thimerosal, containing 49.9% ethyl mercury by weight.

All forms of mercury are well known to be neurotoxic, especially

during early brain development (Costa et al., 2004). The high

affinity binding of mercuric compounds to the thiol (SH) group of

cysteines in essential proteins is thought to be the basis for

mercury-induced cytotoxicity. In vivo studies in rodents have shown

that ethyl mercury is able to cross the cell membrane and then is

converted intracellularly to inorganic mercury which accumulates

preferentially in the brain and kidney (Magos et al., 1985).

Intracellular accumulation of inorganic mercury was shown to be

higher for ethyl compared to methylmercury with equimolar exposure,

although the clearance rate of ethylmercury was faster than

methylmercury (Magos et al., 1985). A recent in vitro study of

Thimerosal exposure to fibroblasts and human cerebral cortical neuron

cell lines demonstrated that short term exposure in concentrations

similar to those used in the present study induced DNA strand breaks,

membrane damage, caspase-3 activation, and cell death (Baskin et al.,

2003). The purpose of the present study was to determine whether the

mechanism of ethylmercury toxicity was similar to that previously

reported for methylmercury. Human glioblastoma and neuroblastoma cell

lines were used as surrogates for astrocytes and neurons,

respectively, based on a previous study demonstrating similar dose-

response profiles to methyl mercury between primary cultures and

transformed cell lines (Sanfeliu et al., 2001).

Glutathione provides the major intracellular defense against ROS and

oxidative stress-induced cell damage and apoptosis (Meister, 1995).

Agents or conditions that deplete mitochondrial glutathione will

indirectly increase ROS levels and induce cell death in a variety of

cell types ( et al., 2001 and Marchetti et al., 1997). Mercury

and other heavy metals are well known to increase oxidative stress

and deplete intracellular glutathione (Naganuma et al., 1990). A

major unanswered question is whether mercury-induced depletion of

glutathione precedes the increase in ROS or whether mercury-induced

ROS induces glutathione depletion. In thymocytes, mitochondrial

glutathione depletion was shown to precede the increase in ROS

associated with loss of viability and apoptosis (Macho et al., 1997).

Whether mercury-induced depletion of glutathione is the initiating

factor for increased oxidative stress and cell death in brain cells

has not yet been evaluated.

A recent in vitro study of Thimerosal immunotoxicity using

immortalized Jurkat T cells demonstrated an increase in reactive

oxygen species and a decrease in intracellular glutathione with

increasing concentrations of Thimerosal (Makani et al., 2002).

Thimerosal, but not thiosalacylic acid (the non-mercury component of

Thimerosal), induced apoptotic cell death in T cells in a

concentration-dependent manner as evidenced by mitochondrial release

of cytochrome c, apoptosis activating factor, and activation of

caspases 9 and 3. Exogenous glutathione inhibited activation of these

caspases and prevented cell death. These results suggest that, at

least in T cells, Thimerosal induces oxidative stress and apoptosis

by activating mitochondrial cell death pathways. A subsequent study

using cultured human neuron and fibroblast cell lines similarly

showed that low micromolar concentrations of Thimerosal induced DNA

strand breaks, caspase-3 activation, membrane damage and cell death

(Baskin et al., 2003).

In the present study, we evaluated glioblastoma cells and

neuroblastoma cells in culture to determine the relative sensitivity

of each cell type to Thimerosal-induced oxidative stress and cell

death. At equimolar concentrations of Thimerosal, the neurons were

found to be much more sensitive to Thimerosal-induced cell death than

the astrocytes. In the neuronal cell line, viability was

significantly reduced in a concentration-dependent manner at 2.5, 5,

10, and 20 & #956;mol/L Thimerosal after only a 3 h exposure, whereas the

astrocytes required a full 48 h exposure for a similar loss of

viability (Fig. 3). These results duplicate observations in the same

cell lines exposed to similar concentrations of methyl mercury and

suggest that the mechanism of ethyl- and methylmercury neurotoxicity

is similar. The addition of either N-acetylcysteine or gluthatione

ethyl ester (100 & #956;mol/L) to the culture medium 1 h before adding 15

& #956;mol/L Thimerosal conferred significant protection against

cytotoxicity in both cell lines (Fig. 4). It is likely that the

extracellular NAC and glutathione provided partial protection by

complexing with the Thimerosal in the culture medium as well as by

increasing intracellular glutathione levels. The oxidized form of

cysteine (cystine) was protective in astrocytes, but not neurons,

consistent with facilitated membrane transport of cystine in

astrocytes (Kranich et al., 1998). Neurons depend on glutathione

synthesized in the astrocytes and released extracellularly where it

is hydrolyzed to cysteinylglycine and cysteine by ectoenzymes to

provide neurons with necessary precursors for intracellular

gluthathione synthesis (Dringen et al., 1999). In both cell lines,

methionine provided no protection against Thimerosal toxicity

confirming the inability of either cell type to synthesize cysteine

(and glutathione) from methionine.

The intracellular concentration of glutathione before supplementation

was 30% lower in neuroblastoma cells compared to the glioblastoma

cells. The lower baseline glutathione concentration in the neuronal

cell line was associated with increased sensitivity to Thimerosal

cytotoxicity (Fig. 5). Thus, sensitivity to Thimerosal was directly

proportional to the basal intracellular glutathione concentration. In

co-culture studies, astrocytes have been shown to protect neurons

against the toxicity of oxidative stress (Dringen et al., 2000a). The

provision of glutathione precursors to neurons is a possible

explanation for the protective effect of astrocytes. Recent results

have confirmed the primary role of astrocytes in glutathione

metabolism and antioxidant defense in the brain (Dringen, 2000).

Depletion of astrocyte glutathione would therefore indirectly induce

oxidative cell death in neurons by depletion of essential glutathione

precursors.

In summary, we have shown that human glioblastoma cells are more

resistant to Thimerosal cytotoxicity than neuroblastoma cells at

doses in the low micromolar range and that the resistance is

correlated with higher intracellular levels of intracellular

glutathione. The significant protection by NAC and glutathione ethyl

ester against Thimerosal cytotoxicity suggests the possibility that

supplementation with glutathione precursors might be protective

against mercury exposures in vivo. Numerous clinical studies have

demonstrated the efficacy of NAC in increasing intracellular

glutathione levels and reducing oxidative stress in humans (

and Luo, 1998 and Badaloo et al., 2002). Since cytotoxicity with both

ethyl- and methylmercury have been shown to be mediated by

glutathione depletion, dietary supplements that increase

intracellular glutathione could be envisioned as an effective

intervention to reduce previous or anticipated exposure to mercury.

This approach would be especially valuable in the elderly and in

pregnant women before receiving flu vaccinations, in pregnant women

receiving Rho D immunoglobulin shots, and individuals who regularly

consume mercury-containing fish.

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Glutathione metabolism in brain metabolic interaction between

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Glutathione pathways in the brain, Biol Chem 384 (2003) (4), pp. 505-

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Corresponding author. Tel.: +1 501 364 4665; fax: +1 501 364 5107.

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>

>

> Theresa:

>

> I agree with you, the EWG report seems remarkable. How we can find

> out more about the protocol that Dr. followed.

>

> It would be completely amazing, not short of a miracle if our

> children could be cured of apraxia...

>

> Does anyone in our group have more information regarding this EWG

> report and the protocol? I'm up for discussion on this important

> issue.

>

> G.-- Do you have any insight regarding the EWG Report and the

> protocol? What are your thoughts?

>

> Thanks,

>

>

>

>

> ,

[Guest guest]

Colleen,

I also am very appreciative of the information,I am gearing up to further

restrict the diet,we had already eliminated artificials but still occasionally

see red ears,dark circles and sleep problems,so perhaps it is the chocolate or

corn

I think we are probably dealing with yeast and I know there is a viral link for

my dd,she keeps getting a red throat off and on and has ticks which come and go

again many thanks for your kind sharing

theresa

[ ] Re:EWG Report ---- Autism report news release

12.13.04

Colleen,

Thanks so much for posting this information! Did you supplement methyl b12

orally or through injections?

Dina

In a message dated 12/28/2004 1:24:03 PM Eastern Standard Time,

writes:

Month 3 we added methyl b 12 every other day

[Guest guest]

Theresa,

Here is some more you may want to take along with you.

Good Luck

Colleen

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PubMed Citation

Articles by , S J.

Articles by Neubrander, J. A

American Journal of Clinical Nutrition, Vol. 80, No. 6, 1611-1617,

December 2004

© 2004 American Society for Clinical Nutrition

----------------------------------------------------------------------

----------

ORIGINAL RESEARCH COMMUNICATION

Metabolic biomarkers of increased oxidative stress and impaired

methylation capacity in children with autism1,2

S Jill , Cutler, Stepan Melnyk, Stefanie Jernigan, Laurette

Janak, W Gaylor and A Neubrander

1 From the Department of Pediatrics, University of Arkansas for

Medical Sciences, and the Arkansas Children's Hospital Research

Institute, Little Rock, AR (SJJ, SM, and SJ); Niagara Falls, NY (PC);

Colden, NY (LJ); Gaylor and Associates, LLC, Eureka Springs, AR

(DWG); and Edison, NJ (JAN)

Background: Autism is a complex neurodevelopmental disorder that

usually presents in early childhood and that is thought to be

influenced by genetic and environmental factors. Although abnormal

metabolism of methionine and homocysteine has been associated with

other neurologic diseases, these pathways have not been evaluated in

persons with autism.

Objective: The purpose of this study was to evaluate plasma

concentrations of metabolites in the methionine transmethylation and

transsulfuration pathways in children diagnosed with autism.

Design: Plasma concentrations of methionine, S-adenosylmethionine

(SAM), S-adenosylhomocysteine (SAH), adenosine, homocysteine,

cystathionine, cysteine, and oxidized and reduced glutathione were

measured in 20 children with autism and in 33 control children. On

the basis of the abnormal metabolic profile, a targeted nutritional

intervention trial with folinic acid, betaine, and methylcobalamin

was initiated in a subset of the autistic children.

Results: Relative to the control children, the children with autism

had significantly lower baseline plasma concentrations of methionine,

SAM, homocysteine, cystathionine, cysteine, and total glutathione and

significantly higher concentrations of SAH, adenosine, and oxidized

glutathione. This metabolic profile is consistent with impaired

capacity for methylation (significantly lower ratio of SAM to SAH)

and increased oxidative stress (significantly lower redox ratio of

reduced glutathione to oxidized glutathione) in children with autism.

The intervention trial was effective in normalizing the metabolic

imbalance in the autistic children.

Conclusions: An increased vulnerability to oxidative stress and a

decreased capacity for methylation may contribute to the development

and clinical manifestation of autism.

Key Words: Autistic disorder • biomarkers • oxidative stress •

methylation • methionine • S-adenosylmethionine • S-

adenosylhomocysteine • adenosine • cysteine • glutathione

----------------------------------------------------------------------

----------

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

Copyright © 2004 by The American Society for Clinical Nutrition, Inc.

> >

> >

> > Theresa:

> >

> > I agree with you, the EWG report seems remarkable. How we can

find

> > out more about the protocol that Dr. followed.

> >

> > It would be completely amazing, not short of a miracle if our

> > children could be cured of apraxia...

> >

> > Does anyone in our group have more information regarding this

EWG

> > report and the protocol? I'm up for discussion on this

important

> > issue.

> >

> > G.-- Do you have any insight regarding the EWG Report and

the

> > protocol? What are your thoughts?

> >

> > Thanks,

> >

> >

> >

> >

> > ,

>

>

>

>

>

>

>

>

>