Study suggests mercury-autism link

Posted on Mon, Dec. 13, 2004

http://www.montereyherald.com/mld/mo...s/10405529.htm



Study links autism to toxic metals

By SANDY KLEFFMAN

Contra Costa Times


A new study sheds light on the mystery of autism and may point the way
to a promising treatment.

Some autistic children have a weakened ability to protect themselves
from toxic metals in their bodies, a biochemist at the University of
Arkansas for Medical Sciences has concluded.

Such children have a severe deficiency of glutathione, the body's most
important tool for detoxifying and excreting heavy metals such as
mercury and lead, Dr. Jill James reports in a peer-reviewed study
published this month in the American Journal of Clinical Nutrition.

James' findings provide new ammunition for those who suspect that
mercury-containing vaccines play a role in triggering autism.

The study, which involved 20 autistic children, also suggests a
possible intervention for the disorder, which has no known cause or
cure.

In an attempt to correct their metabolic imbalance, James gave eight
of the participants supplements of folinic acid, a form of folic acid,
and vitamin B-12. Their glutathione measurements then improved.

The study did not attempt to quantify changes in autistic behavior.

But Dr. Elizabeth Mumper, an associate professor of clinical
pediatrics at the University of Virginia Medical School, said she has
given similar supplements to many autistic children and noticed a
marked improvement in some.

''I don't mean to imply that I can cure autism but for a subset, the
results can be dramatic,'' Mumper said.

Mumper and James said they hope other researchers will attempt to
replicate their findings in larger numbers of children.

Most researchers, including James, say there is a strong genetic
component to autism.

The text of the original paper is pasted below. No where is "mercury"
or "thimerosal" mentioned. What's the connection to mercury? None. How
about reading the original paper BEFORE making such grand statements as
"Study suggests mercury-autism link"?

=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3 D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D
American Journal of Clinical Nutrition, Vol. 80, No. 6, 1611-1617,
December 2004
=A9 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 James, Paul Cutler, Stepan Melnyk, Stefanie Jernigan, Laurette
Janak, David W Gaylor and James 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)

2 Reprints not available. Address correspondence to SJ James, Arkansas
Children's Hospital Research Institute, Slot 512-40B, 1120 Marshall
Street, Little Rock, AR 72202. E-mail: .

ABSTRACT

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 =B7 biomarkers =B7 oxidative stress =B7
methylation =B7 methionine =B7 S-adenosylmethionine =B7
S-adenosylhomocysteine =B7 adenosine =B7 cysteine =B7 glutathione


INTRODUCTION

Autism is a neurodevelopmental disability that is usually diagnosed
before age 3 y and is characterized by deficits in social reciprocity
and in language skills that are associated with repetitive behaviors
and restricted interests (1). In addition to behavioral impairment,
autistic persons have a high prevalence of gastrointestinal disease and
dysbiosis (2), autoimmune disease (3), and mental retardation (4).
Autism also affects many more males than females, occurring at a ratio
of 4:1. A significant role for genetics in the etiology of the autistic
disorder is supported by a high concordance of autism between
monozygotic twins and increased risks among siblings of affected
children and of autistic symptoms associated with several heritable
genetic diseases [see: Online Mendelian Inheritance in Man (OMIM)
#209850 (autism; 5)]. Autism has been reported to be a comorbid
condition associated with Rett syndrome (5), fragile X (6),
phenylketonurea (7), adenylosuccinate lyase deficiency (8),
dihydropyrimidine dehydrogenase deficiency (9), and 5'-nucleotidase
hyperactivity (10); however, these genetic diseases account for 10% of
cases of autism. Nonetheless, the association of autism with genetic
deficits in specific enzymes suggests the possibility that the genetic
component of primary autism could be expressed as a chronic metabolic
imbalance that impairs normal neurodevelopment and immunologic
function. The possibility that autism has a metabolic phenotype is less
widely accepted but has been supported by several small studies (9,
11-14).

The current study was prompted by the serendipitous observation in a
previous study that the metabolic profiles of dizygotic twins-one
with Down syndrome and one with autism-were virtually identical with
respect to methionine cycle and transsulfuration metabolites (15). Down
syndrome, or trisomy 21, is a complex genetic and metabolic disease due
to the presence of 3 copies of chromosome 21 and associated with an
increased frequency of autism (16). In our previous study, children
with Down syndrome had lower concentrations of metabolites in the
methionine cycle and significantly lower glutathione concentrations
than did control children (15).

The methionine cycle involves the regeneration of methionine via the
vitamin B-12-dependent transfer of a methyl group from
5-methyltetrahydrofolate to homocysteine in the methionine synthase
reaction. Methionine may then be activated by methionine
adenosyltransferase to form S-adenosylmethionine (SAM), the primary
methyl donor for most cellular methytransferase reactions including the
methylation of DNA, RNA, proteins, phospholipids, and neurotransmitters
(Figure 1). The transfer of the methyl group from SAM to the various
enzyme-specific methyl acceptors results in the formation of
S-adenosylhomocysteine (SAH). The reversible hydrolysis of SAH to
homocysteine and adenosine by the SAH hydrolase (SAHH) reaction
completes the methionine cycle. Adenosine is further metabolized by
adenosine kinase for purine synthesis or catabolized by adenosine
deaminase. Homocysteine can be either remethylated to methionine or
irreversibly removed from the methionine cycle by cystathionine
=DF-synthase (CBS; 17). Two important consequences of a decrease in
methionine cycle turnover are decreased synthesis of SAM for normal
methylation activity and decreased synthesis of cysteine and
glutathione for normal antioxidant activity.

FIGURE 1. The methionine cycle involves the remethylation of
homocysteine to methionine by either the folate-vitamin
B-12-dependent methionine synthase (MS) reaction or the
folate-vitamin B-12-independent betaine homocysteine
methyltransferase (BHMT) reaction. Methionine is then activated by
methionine adenosyltransferase (MAT) to S-adenosylmethionine (SAM), the
major methyl donor for cellular methyltransferase (MTase) reactions.
After methyl group transfer, SAM is converted to S-adenosylhomocysteine
(SAH), which is further metabolized in a reversible reaction to
homocysteine and adenosine. Adenosine may be phosphorylated to
adenosine nucleotides by adenosine kinase (AK) or catabolized to
inosine by adenosine deaminase (ADA). Homocysteine may be permanently
removed from the methionine cycle by irreversible conversion to
cystathionine by vitamin B-6-dependent cystathionine =DF-synthase
(CBS). Cystathionine is converted to cysteine, which is the
rate-limiting amino acid for the synthesis of the tripeptide
glutathione (Glu-Cys-Gly). THF, tetrohydrofolate; 5-CH3 THF,
5-methyltetrahydrofolate; SAHH, SAH hydrolase.

SUBJECTS AND METHODS
Study participants
The participants in the metabolic study were 20 autistic ( =B1 SD age:
6=2E4 =B1 1.5 y) and 33 control (age: 7.4 =B1 1.3 y) children. The
diagnosis of autism was based on the criteria for autistic disorder as
defined in the Diagnostic and Statistical Manual of Mental Disorders,
Fourth Edition (DSM-IV) and by a diagnostic interview conducted by a
developmental pediatrician. Of the 20 autistic children, all were
white, 14 were boys, 6 were girls, 19 were diagnosed with regressive
autism, and 1 had infantile autism. Most of these children were
impaired in speech and socialization skills and exhibited symptoms of
gastrointestinal distress; before the study, 16 were taking a
multivitamin and mineral supplement containing 400 =B5g folic acid and 3
=B5g vitamin B-12. None of the autistic children were taking prescribed
medicines, such as valproic acid or anticonvulsants, that might have
affected methionine metabolism. A quantifiable diet questionnaire was
not administered as part of this study; thus, specific dietary
differences within and between groups cannot be determined. The control
subjects in the metabolic study were healthy white US children with no
history of chronic disease or autism who had participated in a similar
baseline study of children with Down syndrome (15). Control children
took over-the-counter vitamin supplements and were not taking
medications known to interfere with methionine metabolism. Exclusion
criteria for both groups included a diagnosis of malnutrition, the
presence of active infection, or known genetic disease.

The protocol and informed consent for this study were reviewed and
approved by the Institutional Review Board at the University of
Arkansas for Medical Sciences. The details of the study were explained
to the parents of the participating children, and written informed
consent was obtained from the parents.

Study design
The metabolic study consisted of 3 parts. In the first component,
baseline concentrations of plasma metabolites in the methionine cycle
and transsulfuration pathway were measured in 20 autistic children and
compared with plasma concentrations in 33 control children to establish
whether the metabolic profile of the autistic children differed
significantly from that of the control children. In the second
component, based on the observed abnormalities in plasma metabolites, a
subset of 8 autistic children were given oral supplements of 800 =B5g
folinic acid and 1000 mg betaine (anhydrous trimethylglycine) twice a
day in an attempt to improve the metabolic profile; this is referred to
as intervention 1. After 3 mo on this regimen, blood samples were again
taken and the metabolite concentrations were compared with baseline
concentrations of each metabolite. In the third component, the same
subset of 8 children were given an injectible form of methylcobalamin
(75 =B5g/kg) twice a week in addition to the oral folinic acid and
betaine for an additional month; this is referred to as intervention 2.
Each child served as his or her own control for the intervention study.

Nutritional supplements
USP-grade folinic acid was obtained from Douglas Laboratories
(Pittsburgh) or Thorne Research, Inc (Dover, ID) and was given twice a
day as 800 =B5g oral powder in juice. Betaine (trimethylglycine, USP
grade) was purchased from Life Extension Foundation (Fort Lauderdale,
FL) and given twice a day as 1000 =B5g oral powder in juice. USP
methylcobalamin was obtained from Hopewell Pharmaceuticals (Hopewell,
NJ) or Unique Pharmaceuticals (Temple, TX) as an injectible liquid and
given subcutaneously at a dose of 75 =B5g/kg twice a week.

Sample treatment and HPLC method
Fasting blood samples were collected into EDTA-containing evacuated
tubes (B-D Biosciences, Dallas) and immediately chilled on ice before
being centrifuged at 4000 x g for 10 min at 4 =B0C. Plasma aliquots were
transferred into cryostat tubes and stored at -80 =B0C until
extraction and HPLC quantification. For determination of methionine,
total homocysteine, cysteine, and total glutathione (tGSH)
concentrations, 50 =B5L of a freshly prepared solution of 1.43 mmol
sodium borohydride/L containing 1.5 =B5mol EDTA/L, 66 mmol NaOH/L, and
10 =B5L isoamyl alcohol was added to 200 =B5L plasma to reduce all
sulfhydryl bonds. The samples were incubated at 40 =B0C in a shaker for
30 min. To precipitate proteins, 250 =B5L ice-cold 10% metaphosphoric
acid was added and mixed well, and the sample was incubated for an
additional 10 min on ice. After centrifugation at 18 000 x g for 15 min
at 4 =B0C, the supernatant fluid was filtered through an 0.2-=B5m nylon
membrane filter (PGC Scientific, Frederic, MD), and a 20-=B5L aliquot
was injected into the HPLC system. For measurement of SAM, SAH,
adenosine, cystathionine, and oxidized glutathione (GSSG)
concentrations, 100 =B5L of 10% metaphosphoric acid was added to 200 =B5L
plasma to precipitate protein; the solution was mixed well and
incubated on ice for 30 min. After centrifugation for 15 min at 18 000
g at 4 =B0C, supernatant fluids were passed through an 0.2-=B5m nylon
membrane filter, and 20 =B5L was injected into the HPLC system.

The details of the method for HPLC elution and electrochemical
detection were described previously (18, 19). The separation of
metabolites was performed by using HPLC with a Shimadzu solvent
delivery system (ESA model 580) and a reverse-phase 5-=B5m C18 column
(4.6 x 150 mm; MCM Inc, Tokyo) obtained from ESA Inc (Chemsford, MA). A
20-=B5L aliquot of plasma extract was directly injected onto the column
by using a Beckman Autosampler (model 507E; Beckman Instruments,
Irvine, CA). All plasma metabolites were quantified by using model
5200A Coulochem II and CoulArray electrochemical detection systems
equipped with a dual analytic cell (model 5010), a 4-channel analytic
cell (model 6210), and a guard cell (model 5020) (all: ESA Inc). The
unknown concentrations of plasma metabolites were calculated from peak
areas and standard calibration curves with the use of HPLC software.

Statistical analysis
Metabolic data are presented as means =B1 SDs. Statistical differences
in plasma metabolites between case and control children were
ascertained by using the Student's t test with significance set at
0=2E05. One-way analysis of variance was performed to ascertain whether
differences existed between plasma metabolite concentrations at the 3
time points: baseline (no intervention), after intervention 1 (folinic
acid and betaine), and after intervention 2 (folinic acid, betaine, and
methylcobalamin). Individual metabolites at baseline were subsequently
compared with those after intervention 1 and intervention 2 by using
the paired Student's t test with the Bonferroni correction. Statistical
analyses were accomplished with the use of SIGMASTAT software (version
2=2E0; Systat Software Inc, Richmond. CA).

RESULTS

Baseline methionine cycle and transsulfuration pathway metabolites
The baseline concentrations of metabolites in the methionine cycle and
in the transsulfuration pathway were significantly different between
the autistic children and the control children. Within the methionine
cycle, plasma concentrations of methionine, SAM, and homocysteine were
significantly lower and SAH and adenosine concentrations were
significantly higher than those in the control children (Table 1). The
ratio of SAM to SAH was almost 50% lower in the autistic children than
in the control children. The significant reductions in plasma
cystathionine and cysteine concentrations observed in the autistic
children (Table 1) were consistent with a decrease in CBS-mediated
transsulfuration. Associated with the low mean plasma cysteine
concentration was a significant decrease in tGSH concentrations. GSSG
was increased almost twofold, and tGSH:GSSG was reduced by 70%.


TABLE 1 Comparison of methionine cycle and transsulfuration
metabolites between autistic children and control children1

Supplementation with folinic acid and betaine (intervention 1)
A subset of 8 autistic children participated in an intervention trial
designed to improve their metabolic profile. Oral supplementation with
800 =B5g folinic acid and 1000 mg betaine, both given twice a day, was
maintained for a period of 3 mo (intervention 1), and a second blood
sample was drawn. Relative to baseline concentrations, mean plasma
methionine, SAM, homocysteine, cystathionine, cysteine, and tGSH
concentrations and SAM:SAH and tGSH:GSSG in these 8 children were
higher (Table 2). In addition, the high SAH and adenosine
concentrations observed at baseline decreased with the betaine and
folinic acid supplements during intervention 1. The mean concentrations
of methionine, SAM, SAH, adenosine, and homocysteine were not
statistically different from those in the control children, which
indicated that intervention with folinic acid and betaine had brought
these methionine cycle metabolites into the normal range. Although
supplementation was effective in normalizing the methionine cycle
metabolites to the concentrations in the control subjects, the
intervention significantly improved but did not normalize tGSH or GSSG
concentrations or tGSH:GSSG.


Supplementation with folinic acid, betaine, and methyl vitamin B-12
(intervention 2)
For intervention 2, an injectible form of methylcobalamin (75 =B5g/kg)
was added to the folinic acid and betaine regimen for a period of 1 mo,
after which the third blood sample was taken for HPLC analysis. The
addition of injectible methylcobalamin (intervention 2) did not alter
the mean concentrations of methionine, SAM, SAH, or homocysteine beyond
the alterations induced by the intervention with folinic acid and
betaine (Table 2). However, relative to intervention 1, the addition of
injectible methylcobalamin further decreased the concentrations of
adenosine and GSSG and further increased the concentrations of
methionine, cysteine, and tGSH and SAM:SAH and tGSH:GSSG.


DISCUSSION

Autism is a complex neurodevelopmental disorder that is thought to
involve an interaction between multiple, variable susceptibility genes
(21), epigenetic effects (22), and environmental factors (23). The
apparent increase in the diagnosis of autistic-spectrum disorders from
4-5 in 10 000 children in the 1980s to 30-60 in 10 000 children in
the 1990s has raised great concern (24-27). This increased prevalence
of autism has enormous future public health implications and has
stimulated intense research into potential etiologic factors and
candidate genes. Because abnormal folate metabolism and low glutathione
concentrations have been reported in other neurologic disorders,
including Alzheimer disease, Parkinson disease, schizophrenia, and Down
syndrome (15, 28-31), we measured the concentrations of methionine
methylation and transsulfuration metabolites in a cohort of autistic
children.

The concentrations of metabolites among the control children in this
study were within the range of values previously found in several
studies (32-34). The observed imbalance in methionine and
homocysteine metabolism in the autistic children is complex and not
easily explained by perturbation of a single pathway or isolated
genetic or nutritional deficiency. Within the methionine cycle,
significant decreases in plasma concentrations of methionine, SAM, and
homocysteine were associated with significant increases in adenosine
and SAH. The low methionine and SAM concentrations would suggest a
reduction in methionine synthase activity; however, the observed
decrease in homocysteine does not fit that interpretation. The data may
be best explained by oxidative inactivation of methionine synthase in
combination with a decrease in SAH hydrolase activity secondary to the
increase in adenosine (35, 36). Adenosine binds to the active site of
SAH hydrolase, and increased concentrations of adenosine have been
shown to reduce SAHH activity (36, 37). A combined enzyme deficit would
also be consistent with the observed decrease in SAM and increase in
SAH concentrations. In this case, the decrease in homocysteine
concentrations would reflect an adenosine-mediated decrease in SAH
hydrolysis and homocysteine synthesis. The functional consequence of an
increase in SAH is product inhibition of most cellular
methyltransferases (38). Low methionine and SAM concentrations in
combination with increased SAH and adenosine concentrations were shown
previously to be associated with reduced cellular methylation capacity
(39). The twofold decrease in SAM:SAH also suggests an impaired
capacity in these autistic children for cellular methylation.

The metabolic pattern observed in the transsulfuration pathway may
provide a more cohesive explanation for the unusual imbalance in
methionine cycle metabolites. Low concentrations of cystathionine,
cysteine, and tGSH are consistent with reduced flux through the
transsulfuration pathway. Furthermore, the significant increase in GSSG
disulfide and the 67% decrease in tGSH:GSSG indicate chronic oxidative
stress. Within the methionine cycle, methionine synthase, betaine
homocysteine methyltransferase, and methionine adenosyltransferase are
all redox-sensitive enzymes that are down-regulated by oxidative stress
(40-42). A decrease in methionine- and SAM-regulated CBS activity
would increase the requirement for cysteine, effectively making it an
essential amino acid in these children. Because cysteine is the
rate-limiting amino acid for glutathione synthesis, its decrease is
consistent with low concentrations of glutathione (43, 44). The
remarkably consistent decrease in cysteine and glutathione
concentrations and tGSH:GSSG in the autistic children suggests an
increased vulnerability to oxidative stress.

The genetic or environmental factors (or both) that would initiate
oxidative stress and abnormal metabolic profiles in the autistic
children are not clear. It is possibly relevant that, in autistic
children, decreased activity of adenosine deaminase and increased
frequency of adenosine deaminase polymorphisms have been shown to be
associated with low adenosine deaminase activity (14, 45). The observed
increase in adenosine could be due to either an inhibition of adenosine
kinase or an increase in 5-nucleotidase, both of which have been shown
to occur with oxidative stress (46, 47). Elevated intracellular
adenosine has been shown to inhibit glutathione synthesis (48, 49).
Alternatively, a genetic predisposition to environmental agents or
conditions that promote oxidative stress could contribute to the
abnormal metabolic profile observed in the autistic children.

The targeted nutritional intervention trial in a subset of the autistic
children was specifically designed to increase methionine
concentrations (intervention 1). Betaine homocysteine methyltransferase
provides a folate-vitamin B-12-independent pathway in the liver and
kidney to remethylate homocysteine to methionine (17). Supplemental
betaine (trimethylglycine) has been shown to up-regulate betaine
homocysteine methyltransferase expression and activity to increase
methionine synthesis (50). Folinic acid (5-formyl tetrahydrofolate) was
used rather than folic acid because the former is absorbed as the
reduced metabolite and can enter folate metabolism as 5,10-methylene
tetrahydrofolate, thereby reducing the possibility of promoting a
folate trap (51, 52). As shown in Table 2, the intervention with
betaine and folinic acid was successful in bringing all the metabolites
within the methionine cycle into the normal range and simultaneously
improving significantly the metabolites in the transsulfuration
pathway. The increase in methionine, SAM, and homocysteine
concentrations and the decrease in adenosine and SAH concentrations
suggested that the intervention stimulated an increased flux through
the methionine cycle. In addition, the significant increase in
cystathionine concentrations suggests that the supplements were
effective also in increasing CBS activity, most likely because of
up-regulation by the increase in SAM. The associated increases in
cysteine and glutathione indicate that transsulfuration to glutathione
was enhanced by the supplements. The decrease in adenosine is
consistent with a concomitant release of SAHH inhibition and decrease
in SAH and, possibly, the release of a bottleneck in methionine cycle
turnover. The mechanism for the decrease in adenosine concentrations,
however, is not clear. One possibility is that the increase in cysteine
or glutathione concentration (or both) relieved the need for adenosine
as a protective factor against oxidative damage (53, 54).

The addition of injectible methylcobalamin to the protocol
(intervention 2) was based on empirical observations of clinical
improvement in speech and cognition (by JAN) and the possibility that
it might enhance methionine synthase activity under conditions of
oxidative stress by replacing oxidized inactive coenzyme B-12
[cob(II)alamin] or by posttranslational up-regulation of methionine
synthase, or both (55, 56). One month after the addition of
methylcobalamin, the methionine concentrations were within the control
range (Table 1), and further improvements in adenosine and SAH
concentrations and SAM:SAH were observed. Unexpectedly, and perhaps
most significantly, the addition of methylcobalamin reduced the
concentrations of inactive GSSG and increased the tGSH concentrations
and tGSH:GSSG so that they were not different from those in the control
children (Table 1). These positive changes in the glutathione redox
profile most likely reflect the increase in cysteine as the
rate-limiting amino acid for glutathione synthesis (44). Of note, there
is a higher demand for cysteine (and, indirectly, methionine) for de
novo glutathione synthesis during chronic oxidative stress (43). Low
antioxidant enzyme activity in autistic children has been reported in 3
recent studies (57-59) that provide additional support for oxidative
stress as a part of the etiology of autism. If the decreases in plasma
methionine, cysteine, and glutathione concentrations in autistic
children observed in the current study are confirmed in a larger study,
low concentrations of these thiol metabolites could provide metabolic
biomarkers for autism.

Although clinical improvements in speech and cognition were noted by
the attending physician (PC), they were not measured in a quantifiable
manner and are therefore not reported here. Specific dietary
differences between groups could have contributed to our results, but
we consider it unlikely that uniform dietary differences within the
autistic group existed that could have accounted for the remarkably
consistent metabolic alterations. Increased frequency of common
polymorphisms in these pathways may have contributed to the observed
metabolic phenotype, and studies of that subject, as well as studies to
quantify clinical improvement, are currently underway. Our attempts to
interpret these preliminary metabolic findings are clearly speculative,
and a better understanding of the abnormal one-carbon metabolism in
these children will require additional research efforts. Nonetheless,
the ability to correct the metabolic imbalance with targeted
nutritional intervention implies that certain aspects of autism may be
treatable.

Nineteen of the 20 children participating in the study were diagnosed
with "regressive" autism (apparently normal development until
regression into autism between ages 1.5 and 3 y). On the basis of their
abnormal metabolic profiles, we hypothesize that an increased
vulnerability to oxidative stress (environmental, intracellular, or
both) and impaired methylation capacity may contribute to the
development and clinical manifestation of regressive autism.


ACKNOWLEDGMENTS

SJJ was responsible for study design, study coordination,
interpretation of data, and manuscript writing. PC was responsible for
patient recruitment, obtaining supplements, patient compliance,
monitoring clinical status, and methylcobalamin injections. SM was
responsible for HPLC quantification of plasma metabolites and data
collection and interpretation. SJ was responsible for plasma and DNA
extraction and data collection and interpretation. LJ was responsible
for patient recruitment, study coordinating and consulting, and data
interpretation. DWG was responsible for statistical analysis of data.
JAN was responsible for initiating the methylcobalamin treatment in
autistic patients, providing consultation, and interpreting data. None
of the authors has any financial conflict of interest.


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Received for publication June 1, 2004. Accepted for publication August
23, 2004.

Could you please show us the peer-reviewed research papers?

Thanks.

Jeff

"Jeff" wrote in message
...
Could you please show us the peer-reviewed research papers?

"Dr. Jill James reports in a peer-reviewed study published this month in the
American Journal of Clinical Nutrition"

On Tue, 14 Dec 2004 21:35:42 -0500, "Jeff"
wrote:

Could you please show us the peer-reviewed research papers?


LOL ... you mean by "peer reviewers" like Quack Barrett is????

Thank God you no longer have ANY medical license ... not even your
restricted one ... how long has it been, Jeffie?

7 years or more already?

The text of the original paper is pasted below. No where are "mercury"
or "thimerosal" mentioned. What's the connection to mercury? None. No
where is "vaccine" mentioned. What's the connection to vaccines? None.
How about reading the original paper BEFORE making such grand
statements as "Study suggests mercury-autism link"?

=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3 D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D
American Journal of Clinical Nutrition, Vol. 80, No. 6, 1611-1617,
December 2004
=A9 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 James, Paul Cutler, Stepan Melnyk, Stefanie Jernigan, Laurette
Janak, David W Gaylor and James 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)

2 Reprints not available. Address correspondence to SJ James, Arkansas
Children's Hospital Research Institute, Slot 512-40B, 1120 Marshall
Street, Little Rock, AR 72202. E-mail: .

ABSTRACT

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 =B7 biomarkers =B7 oxidative stress =B7
methylation =B7 methionine =B7 S-adenosylmethionine =B7
S-adenosylhomocysteine =B7 adenosine =B7 cysteine =B7 glutathione


INTRODUCTION

Autism is a neurodevelopmental disability that is usually diagnosed
before age 3 y and is characterized by deficits in social reciprocity
and in language skills that are associated with repetitive behaviors
and restricted interests (1). In addition to behavioral impairment,
autistic persons have a high prevalence of gastrointestinal disease and
dysbiosis (2), autoimmune disease (3), and mental retardation (4).
Autism also affects many more males than females, occurring at a ratio
of 4:1. A significant role for genetics in the etiology of the autistic
disorder is supported by a high concordance of autism between
monozygotic twins and increased risks among siblings of affected
children and of autistic symptoms associated with several heritable
genetic diseases [see: Online Mendelian Inheritance in Man (OMIM)
#209850 (autism; 5)]. Autism has been reported to be a comorbid
condition associated with Rett syndrome (5), fragile X (6),
phenylketonurea (7), adenylosuccinate lyase deficiency (8),
dihydropyrimidine dehydrogenase deficiency (9), and 5'-nucleotidase
hyperactivity (10); however, these genetic diseases account for 10% of
cases of autism. Nonetheless, the association of autism with genetic
deficits in specific enzymes suggests the possibility that the genetic
component of primary autism could be expressed as a chronic metabolic
imbalance that impairs normal neurodevelopment and immunologic
function. The possibility that autism has a metabolic phenotype is less
widely accepted but has been supported by several small studies (9,
11-14).

The current study was prompted by the serendipitous observation in a
previous study that the metabolic profiles of dizygotic twins-one
with Down syndrome and one with autism-were virtually identical with
respect to methionine cycle and transsulfuration metabolites (15). Down
syndrome, or trisomy 21, is a complex genetic and metabolic disease due
to the presence of 3 copies of chromosome 21 and associated with an
increased frequency of autism (16). In our previous study, children
with Down syndrome had lower concentrations of metabolites in the
methionine cycle and significantly lower glutathione concentrations
than did control children (15).

The methionine cycle involves the regeneration of methionine via the
vitamin B-12-dependent transfer of a methyl group from
5-methyltetrahydrofolate to homocysteine in the methionine synthase
reaction. Methionine may then be activated by methionine
adenosyltransferase to form S-adenosylmethionine (SAM), the primary
methyl donor for most cellular methytransferase reactions including the
methylation of DNA, RNA, proteins, phospholipids, and neurotransmitters
(Figure 1). The transfer of the methyl group from SAM to the various
enzyme-specific methyl acceptors results in the formation of
S-adenosylhomocysteine (SAH). The reversible hydrolysis of SAH to
homocysteine and adenosine by the SAH hydrolase (SAHH) reaction
completes the methionine cycle. Adenosine is further metabolized by
adenosine kinase for purine synthesis or catabolized by adenosine
deaminase. Homocysteine can be either remethylated to methionine or
irreversibly removed from the methionine cycle by cystathionine
=DF-synthase (CBS; 17). Two important consequences of a decrease in
methionine cycle turnover are decreased synthesis of SAM for normal
methylation activity and decreased synthesis of cysteine and
glutathione for normal antioxidant activity.

FIGURE 1. The methionine cycle involves the remethylation of
homocysteine to methionine by either the folate-vitamin
B-12-dependent methionine synthase (MS) reaction or the
folate-vitamin B-12-independent betaine homocysteine
methyltransferase (BHMT) reaction. Methionine is then activated by
methionine adenosyltransferase (MAT) to S-adenosylmethionine (SAM), the
major methyl donor for cellular methyltransferase (MTase) reactions.
After methyl group transfer, SAM is converted to S-adenosylhomocysteine
(SAH), which is further metabolized in a reversible reaction to
homocysteine and adenosine. Adenosine may be phosphorylated to
adenosine nucleotides by adenosine kinase (AK) or catabolized to
inosine by adenosine deaminase (ADA). Homocysteine may be permanently
removed from the methionine cycle by irreversible conversion to
cystathionine by vitamin B-6-dependent cystathionine =DF-synthase
(CBS). Cystathionine is converted to cysteine, which is the
rate-limiting amino acid for the synthesis of the tripeptide
glutathione (Glu-Cys-Gly). THF, tetrohydrofolate; 5-CH3 THF,
5-methyltetrahydrofolate; SAHH, SAH hydrolase.

SUBJECTS AND METHODS
Study participants
The participants in the metabolic study were 20 autistic ( =B1 SD age:
6=2E4 =B1 1.5 y) and 33 control (age: 7.4 =B1 1.3 y) children. The
diagnosis of autism was based on the criteria for autistic disorder as
defined in the Diagnostic and Statistical Manual of Mental Disorders,
Fourth Edition (DSM-IV) and by a diagnostic interview conducted by a
developmental pediatrician. Of the 20 autistic children, all were
white, 14 were boys, 6 were girls, 19 were diagnosed with regressive
autism, and 1 had infantile autism. Most of these children were
impaired in speech and socialization skills and exhibited symptoms of
gastrointestinal distress; before the study, 16 were taking a
multivitamin and mineral supplement containing 400 =B5g folic acid and 3
=B5g vitamin B-12. None of the autistic children were taking prescribed
medicines, such as valproic acid or anticonvulsants, that might have
affected methionine metabolism. A quantifiable diet questionnaire was
not administered as part of this study; thus, specific dietary
differences within and between groups cannot be determined. The control
subjects in the metabolic study were healthy white US children with no
history of chronic disease or autism who had participated in a similar
baseline study of children with Down syndrome (15). Control children
took over-the-counter vitamin supplements and were not taking
medications known to interfere with methionine metabolism. Exclusion
criteria for both groups included a diagnosis of malnutrition, the
presence of active infection, or known genetic disease.

The protocol and informed consent for this study were reviewed and
approved by the Institutional Review Board at the University of
Arkansas for Medical Sciences. The details of the study were explained
to the parents of the participating children, and written informed
consent was obtained from the parents.

Study design
The metabolic study consisted of 3 parts. In the first component,
baseline concentrations of plasma metabolites in the methionine cycle
and transsulfuration pathway were measured in 20 autistic children and
compared with plasma concentrations in 33 control children to establish
whether the metabolic profile of the autistic children differed
significantly from that of the control children. In the second
component, based on the observed abnormalities in plasma metabolites, a
subset of 8 autistic children were given oral supplements of 800 =B5g
folinic acid and 1000 mg betaine (anhydrous trimethylglycine) twice a
day in an attempt to improve the metabolic profile; this is referred to
as intervention 1. After 3 mo on this regimen, blood samples were again
taken and the metabolite concentrations were compared with baseline
concentrations of each metabolite. In the third component, the same
subset of 8 children were given an injectible form of methylcobalamin
(75 =B5g/kg) twice a week in addition to the oral folinic acid and
betaine for an additional month; this is referred to as intervention 2.
Each child served as his or her own control for the intervention study.

Nutritional supplements
USP-grade folinic acid was obtained from Douglas Laboratories
(Pittsburgh) or Thorne Research, Inc (Dover, ID) and was given twice a
day as 800 =B5g oral powder in juice. Betaine (trimethylglycine, USP
grade) was purchased from Life Extension Foundation (Fort Lauderdale,
FL) and given twice a day as 1000 =B5g oral powder in juice. USP
methylcobalamin was obtained from Hopewell Pharmaceuticals (Hopewell,
NJ) or Unique Pharmaceuticals (Temple, TX) as an injectible liquid and
given subcutaneously at a dose of 75 =B5g/kg twice a week.

Sample treatment and HPLC method
Fasting blood samples were collected into EDTA-containing evacuated
tubes (B-D Biosciences, Dallas) and immediately chilled on ice before
being centrifuged at 4000 x g for 10 min at 4 =B0C. Plasma aliquots were
transferred into cryostat tubes and stored at -80 =B0C until
extraction and HPLC quantification. For determination of methionine,
total homocysteine, cysteine, and total glutathione (tGSH)
concentrations, 50 =B5L of a freshly prepared solution of 1.43 mmol
sodium borohydride/L containing 1.5 =B5mol EDTA/L, 66 mmol NaOH/L, and
10 =B5L isoamyl alcohol was added to 200 =B5L plasma to reduce all
sulfhydryl bonds. The samples were incubated at 40 =B0C in a shaker for
30 min. To precipitate proteins, 250 =B5L ice-cold 10% metaphosphoric
acid was added and mixed well, and the sample was incubated for an
additional 10 min on ice. After centrifugation at 18 000 x g for 15 min
at 4 =B0C, the supernatant fluid was filtered through an 0.2-=B5m nylon
membrane filter (PGC Scientific, Frederic, MD), and a 20-=B5L aliquot
was injected into the HPLC system. For measurement of SAM, SAH,
adenosine, cystathionine, and oxidized glutathione (GSSG)
concentrations, 100 =B5L of 10% metaphosphoric acid was added to 200 =B5L
plasma to precipitate protein; the solution was mixed well and
incubated on ice for 30 min. After centrifugation for 15 min at 18 000
g at 4 =B0C, supernatant fluids were passed through an 0.2-=B5m nylon
membrane filter, and 20 =B5L was injected into the HPLC system.

The details of the method for HPLC elution and electrochemical
detection were described previously (18, 19). The separation of
metabolites was performed by using HPLC with a Shimadzu solvent
delivery system (ESA model 580) and a reverse-phase 5-=B5m C18 column
(4.6 x 150 mm; MCM Inc, Tokyo) obtained from ESA Inc (Chemsford, MA). A
20-=B5L aliquot of plasma extract was directly injected onto the column
by using a Beckman Autosampler (model 507E; Beckman Instruments,
Irvine, CA). All plasma metabolites were quantified by using model
5200A Coulochem II and CoulArray electrochemical detection systems
equipped with a dual analytic cell (model 5010), a 4-channel analytic
cell (model 6210), and a guard cell (model 5020) (all: ESA Inc). The
unknown concentrations of plasma metabolites were calculated from peak
areas and standard calibration curves with the use of HPLC software.

Statistical analysis
Metabolic data are presented as means =B1 SDs. Statistical differences
in plasma metabolites between case and control children were
ascertained by using the Student's t test with significance set at
0=2E05. One-way analysis of variance was performed to ascertain whether
differences existed between plasma metabolite concentrations at the 3
time points: baseline (no intervention), after intervention 1 (folinic
acid and betaine), and after intervention 2 (folinic acid, betaine, and
methylcobalamin). Individual metabolites at baseline were subsequently
compared with those after intervention 1 and intervention 2 by using
the paired Student's t test with the Bonferroni correction. Statistical
analyses were accomplished with the use of SIGMASTAT software (version
2=2E0; Systat Software Inc, Richmond. CA).

RESULTS

Baseline methionine cycle and transsulfuration pathway metabolites
The baseline concentrations of metabolites in the methionine cycle and
in the transsulfuration pathway were significantly different between
the autistic children and the control children. Within the methionine
cycle, plasma concentrations of methionine, SAM, and homocysteine were
significantly lower and SAH and adenosine concentrations were
significantly higher than those in the control children (Table 1). The
ratio of SAM to SAH was almost 50% lower in the autistic children than
in the control children. The significant reductions in plasma
cystathionine and cysteine concentrations observed in the autistic
children (Table 1) were consistent with a decrease in CBS-mediated
transsulfuration. Associated with the low mean plasma cysteine
concentration was a significant decrease in tGSH concentrations. GSSG
was increased almost twofold, and tGSH:GSSG was reduced by 70%.


TABLE 1 Comparison of methionine cycle and transsulfuration
metabolites between autistic children and control children1

Supplementation with folinic acid and betaine (intervention 1)
A subset of 8 autistic children participated in an intervention trial
designed to improve their metabolic profile. Oral supplementation with
800 =B5g folinic acid and 1000 mg betaine, both given twice a day, was
maintained for a period of 3 mo (intervention 1), and a second blood
sample was drawn. Relative to baseline concentrations, mean plasma
methionine, SAM, homocysteine, cystathionine, cysteine, and tGSH
concentrations and SAM:SAH and tGSH:GSSG in these 8 children were
higher (Table 2). In addition, the high SAH and adenosine
concentrations observed at baseline decreased with the betaine and
folinic acid supplements during intervention 1. The mean concentrations
of methionine, SAM, SAH, adenosine, and homocysteine were not
statistically different from those in the control children, which
indicated that intervention with folinic acid and betaine had brought
these methionine cycle metabolites into the normal range. Although
supplementation was effective in normalizing the methionine cycle
metabolites to the concentrations in the control subjects, the
intervention significantly improved but did not normalize tGSH or GSSG
concentrations or tGSH:GSSG.


Supplementation with folinic acid, betaine, and methyl vitamin B-12
(intervention 2)
For intervention 2, an injectible form of methylcobalamin (75 =B5g/kg)
was added to the folinic acid and betaine regimen for a period of 1 mo,
after which the third blood sample was taken for HPLC analysis. The
addition of injectible methylcobalamin (intervention 2) did not alter
the mean concentrations of methionine, SAM, SAH, or homocysteine beyond
the alterations induced by the intervention with folinic acid and
betaine (Table 2). However, relative to intervention 1, the addition of
injectible methylcobalamin further decreased the concentrations of
adenosine and GSSG and further increased the concentrations of
methionine, cysteine, and tGSH and SAM:SAH and tGSH:GSSG.


DISCUSSION

Autism is a complex neurodevelopmental disorder that is thought to
involve an interaction between multiple, variable susceptibility genes
(21), epigenetic effects (22), and environmental factors (23). The
apparent increase in the diagnosis of autistic-spectrum disorders from
4-5 in 10 000 children in the 1980s to 30-60 in 10 000 children in
the 1990s has raised great concern (24-27). This increased prevalence
of autism has enormous future public health implications and has
stimulated intense research into potential etiologic factors and
candidate genes. Because abnormal folate metabolism and low glutathione
concentrations have been reported in other neurologic disorders,
including Alzheimer disease, Parkinson disease, schizophrenia, and Down
syndrome (15, 28-31), we measured the concentrations of methionine
methylation and transsulfuration metabolites in a cohort of autistic
children.

The concentrations of metabolites among the control children in this
study were within the range of values previously found in several
studies (32-34). The observed imbalance in methionine and
homocysteine metabolism in the autistic children is complex and not
easily explained by perturbation of a single pathway or isolated
genetic or nutritional deficiency. Within the methionine cycle,
significant decreases in plasma concentrations of methionine, SAM, and
homocysteine were associated with significant increases in adenosine
and SAH. The low methionine and SAM concentrations would suggest a
reduction in methionine synthase activity; however, the observed
decrease in homocysteine does not fit that interpretation. The data may
be best explained by oxidative inactivation of methionine synthase in
combination with a decrease in SAH hydrolase activity secondary to the
increase in adenosine (35, 36). Adenosine binds to the active site of
SAH hydrolase, and increased concentrations of adenosine have been
shown to reduce SAHH activity (36, 37). A combined enzyme deficit would
also be consistent with the observed decrease in SAM and increase in
SAH concentrations. In this case, the decrease in homocysteine
concentrations would reflect an adenosine-mediated decrease in SAH
hydrolysis and homocysteine synthesis. The functional consequence of an
increase in SAH is product inhibition of most cellular
methyltransferases (38). Low methionine and SAM concentrations in
combination with increased SAH and adenosine concentrations were shown
previously to be associated with reduced cellular methylation capacity
(39). The twofold decrease in SAM:SAH also suggests an impaired
capacity in these autistic children for cellular methylation.

The metabolic pattern observed in the transsulfuration pathway may
provide a more cohesive explanation for the unusual imbalance in
methionine cycle metabolites. Low concentrations of cystathionine,
cysteine, and tGSH are consistent with reduced flux through the
transsulfuration pathway. Furthermore, the significant increase in GSSG
disulfide and the 67% decrease in tGSH:GSSG indicate chronic oxidative
stress. Within the methionine cycle, methionine synthase, betaine
homocysteine methyltransferase, and methionine adenosyltransferase are
all redox-sensitive enzymes that are down-regulated by oxidative stress
(40-42). A decrease in methionine- and SAM-regulated CBS activity
would increase the requirement for cysteine, effectively making it an
essential amino acid in these children. Because cysteine is the
rate-limiting amino acid for glutathione synthesis, its decrease is
consistent with low concentrations of glutathione (43, 44). The
remarkably consistent decrease in cysteine and glutathione
concentrations and tGSH:GSSG in the autistic children suggests an
increased vulnerability to oxidative stress.

The genetic or environmental factors (or both) that would initiate
oxidative stress and abnormal metabolic profiles in the autistic
children are not clear. It is possibly relevant that, in autistic
children, decreased activity of adenosine deaminase and increased
frequency of adenosine deaminase polymorphisms have been shown to be
associated with low adenosine deaminase activity (14, 45). The observed
increase in adenosine could be due to either an inhibition of adenosine
kinase or an increase in 5-nucleotidase, both of which have been shown
to occur with oxidative stress (46, 47). Elevated intracellular
adenosine has been shown to inhibit glutathione synthesis (48, 49).
Alternatively, a genetic predisposition to environmental agents or
conditions that promote oxidative stress could contribute to the
abnormal metabolic profile observed in the autistic children.

The targeted nutritional intervention trial in a subset of the autistic
children was specifically designed to increase methionine
concentrations (intervention 1). Betaine homocysteine methyltransferase
provides a folate-vitamin B-12-independent pathway in the liver and
kidney to remethylate homocysteine to methionine (17). Supplemental
betaine (trimethylglycine) has been shown to up-regulate betaine
homocysteine methyltransferase expression and activity to increase
methionine synthesis (50). Folinic acid (5-formyl tetrahydrofolate) was
used rather than folic acid because the former is absorbed as the
reduced metabolite and can enter folate metabolism as 5,10-methylene
tetrahydrofolate, thereby reducing the possibility of promoting a
folate trap (51, 52). As shown in Table 2, the intervention with
betaine and folinic acid was successful in bringing all the metabolites
within the methionine cycle into the normal range and simultaneously
improving significantly the metabolites in the transsulfuration
pathway. The increase in methionine, SAM, and homocysteine
concentrations and the decrease in adenosine and SAH concentrations
suggested that the intervention stimulated an increased flux through
the methionine cycle. In addition, the significant increase in
cystathionine concentrations suggests that the supplements were
effective also in increasing CBS activity, most likely because of
up-regulation by the increase in SAM. The associated increases in
cysteine and glutathione indicate that transsulfuration to glutathione
was enhanced by the supplements. The decrease in adenosine is
consistent with a concomitant release of SAHH inhibition and decrease
in SAH and, possibly, the release of a bottleneck in methionine cycle
turnover. The mechanism for the decrease in adenosine concentrations,
however, is not clear. One possibility is that the increase in cysteine
or glutathione concentration (or both) relieved the need for adenosine
as a protective factor against oxidative damage (53, 54).

The addition of injectible methylcobalamin to the protocol
(intervention 2) was based on empirical observations of clinical
improvement in speech and cognition (by JAN) and the possibility that
it might enhance methionine synthase activity under conditions of
oxidative stress by replacing oxidized inactive coenzyme B-12
[cob(II)alamin] or by posttranslational up-regulation of methionine
synthase, or both (55, 56). One month after the addition of
methylcobalamin, the methionine concentrations were within the control
range (Table 1), and further improvements in adenosine and SAH
concentrations and SAM:SAH were observed. Unexpectedly, and perhaps
most significantly, the addition of methylcobalamin reduced the
concentrations of inactive GSSG and increased the tGSH concentrations
and tGSH:GSSG so that they were not different from those in the control
children (Table 1). These positive changes in the glutathione redox
profile most likely reflect the increase in cysteine as the
rate-limiting amino acid for glutathione synthesis (44). Of note, there
is a higher demand for cysteine (and, indirectly, methionine) for de
novo glutathione synthesis during chronic oxidative stress (43). Low
antioxidant enzyme activity in autistic children has been reported in 3
recent studies (57-59) that provide additional support for oxidative
stress as a part of the etiology of autism. If the decreases in plasma
methionine, cysteine, and glutathione concentrations in autistic
children observed in the current study are confirmed in a larger study,
low concentrations of these thiol metabolites could provide metabolic
biomarkers for autism.

Although clinical improvements in speech and cognition were noted by
the attending physician (PC), they were not measured in a quantifiable
manner and are therefore not reported here. Specific dietary
differences between groups could have contributed to our results, but
we consider it unlikely that uniform dietary differences within the
autistic group existed that could have accounted for the remarkably
consistent metabolic alterations. Increased frequency of common
polymorphisms in these pathways may have contributed to the observed
metabolic phenotype, and studies of that subject, as well as studies to
quantify clinical improvement, are currently underway. Our attempts to
interpret these preliminary metabolic findings are clearly speculative,
and a better understanding of the abnormal one-carbon metabolism in
these children will require additional research efforts. Nonetheless,
the ability to correct the metabolic imbalance with targeted
nutritional intervention implies that certain aspects of autism may be
treatable.

Nineteen of the 20 children participating in the study were diagnosed
with "regressive" autism (apparently normal development until
regression into autism between ages 1.5 and 3 y). On the basis of their
abnormal metabolic profiles, we hypothesize that an increased
vulnerability to oxidative stress (environmental, intracellular, or
both) and impaired methylation capacity may contribute to the
development and clinical manifestation of regressive autism.


ACKNOWLEDGMENTS

SJJ was responsible for study design, study coordination,
interpretation of data, and manuscript writing. PC was responsible for
patient recruitment, obtaining supplements, patient compliance,
monitoring clinical status, and methylcobalamin injections. SM was
responsible for HPLC quantification of plasma metabolites and data
collection and interpretation. SJ was responsible for plasma and DNA
extraction and data collection and interpretation. LJ was responsible
for patient recruitment, study coordinating and consulting, and data
interpretation. DWG was responsible for statistical analysis of data.
JAN was responsible for initiating the methylcobalamin treatment in
autistic patients, providing consultation, and interpreting data. None
of the authors has any financial conflict of interest.


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Received for publication June 1, 2004. Accepted for publication August
23, 2004.

(Note: this is my 4th attempt at posting this. The new Google News is a
real pain...)

The text of the original paper is pasted below. No where are "mercury"
or "thimerosal" mentioned. What's the connection to mercury? None. How
about reading the original paper BEFORE making such grand statements as
"Study suggests mercury-autism link"?

=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3 D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D
American Journal of Clinical Nutrition, Vol. 80, No. 6, 1611-1617,
December 2004
=A9 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 James, Paul Cutler, Stepan Melnyk, Stefanie Jernigan, Laurette
Janak, David W Gaylor and James 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)

2 Reprints not available. Address correspondence to SJ James, Arkansas
Children's Hospital Research Institute, Slot 512-40B, 1120 Marshall
Street, Little Rock, AR 72202. E-mail: .

ABSTRACT

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 =B7 biomarkers =B7 oxidative stress =B7
methylation =B7 methionine =B7 S-adenosylmethionine =B7
S-adenosylhomocysteine =B7 adenosine =B7 cysteine =B7 glutathione


INTRODUCTION

Autism is a neurodevelopmental disability that is usually diagnosed
before age 3 y and is characterized by deficits in social reciprocity
and in language skills that are associated with repetitive behaviors
and restricted interests (1). In addition to behavioral impairment,
autistic persons have a high prevalence of gastrointestinal disease and
dysbiosis (2), autoimmune disease (3), and mental retardation (4).
Autism also affects many more males than females, occurring at a ratio
of 4:1. A significant role for genetics in the etiology of the autistic
disorder is supported by a high concordance of autism between
monozygotic twins and increased risks among siblings of affected
children and of autistic symptoms associated with several heritable
genetic diseases [see: Online Mendelian Inheritance in Man (OMIM)
#209850 (autism; 5)]. Autism has been reported to be a comorbid
condition associated with Rett syndrome (5), fragile X (6),
phenylketonurea (7), adenylosuccinate lyase deficiency (8),
dihydropyrimidine dehydrogenase deficiency (9), and 5'-nucleotidase
hyperactivity (10); however, these genetic diseases account for 10% of
cases of autism. Nonetheless, the association of autism with genetic
deficits in specific enzymes suggests the possibility that the genetic
component of primary autism could be expressed as a chronic metabolic
imbalance that impairs normal neurodevelopment and immunologic
function. The possibility that autism has a metabolic phenotype is less
widely accepted but has been supported by several small studies (9,
11-14).

The current study was prompted by the serendipitous observation in a
previous study that the metabolic profiles of dizygotic twins-one
with Down syndrome and one with autism-were virtually identical with
respect to methionine cycle and transsulfuration metabolites (15). Down
syndrome, or trisomy 21, is a complex genetic and metabolic disease due
to the presence of 3 copies of chromosome 21 and associated with an
increased frequency of autism (16). In our previous study, children
with Down syndrome had lower concentrations of metabolites in the
methionine cycle and significantly lower glutathione concentrations
than did control children (15).

The methionine cycle involves the regeneration of methionine via the
vitamin B-12-dependent transfer of a methyl group from
5-methyltetrahydrofolate to homocysteine in the methionine synthase
reaction. Methionine may then be activated by methionine
adenosyltransferase to form S-adenosylmethionine (SAM), the primary
methyl donor for most cellular methytransferase reactions including the
methylation of DNA, RNA, proteins, phospholipids, and neurotransmitters
(Figure 1). The transfer of the methyl group from SAM to the various
enzyme-specific methyl acceptors results in the formation of
S-adenosylhomocysteine (SAH). The reversible hydrolysis of SAH to
homocysteine and adenosine by the SAH hydrolase (SAHH) reaction
completes the methionine cycle. Adenosine is further metabolized by
adenosine kinase for purine synthesis or catabolized by adenosine
deaminase. Homocysteine can be either remethylated to methionine or
irreversibly removed from the methionine cycle by cystathionine
=DF-synthase (CBS; 17). Two important consequences of a decrease in
methionine cycle turnover are decreased synthesis of SAM for normal
methylation activity and decreased synthesis of cysteine and
glutathione for normal antioxidant activity.

FIGURE 1. The methionine cycle involves the remethylation of
homocysteine to methionine by either the folate-vitamin
B-12-dependent methionine synthase (MS) reaction or the
folate-vitamin B-12-independent betaine homocysteine
methyltransferase (BHMT) reaction. Methionine is then activated by
methionine adenosyltransferase (MAT) to S-adenosylmethionine (SAM), the
major methyl donor for cellular methyltransferase (MTase) reactions.
After methyl group transfer, SAM is converted to S-adenosylhomocysteine
(SAH), which is further metabolized in a reversible reaction to
homocysteine and adenosine. Adenosine may be phosphorylated to
adenosine nucleotides by adenosine kinase (AK) or catabolized to
inosine by adenosine deaminase (ADA). Homocysteine may be permanently
removed from the methionine cycle by irreversible conversion to
cystathionine by vitamin B-6-dependent cystathionine =DF-synthase
(CBS). Cystathionine is converted to cysteine, which is the
rate-limiting amino acid for the synthesis of the tripeptide
glutathione (Glu-Cys-Gly). THF, tetrohydrofolate; 5-CH3 THF,
5-methyltetrahydrofolate; SAHH, SAH hydrolase.

SUBJECTS AND METHODS
Study participants
The participants in the metabolic study were 20 autistic ( =B1 SD age:
6=2E4 =B1 1.5 y) and 33 control (age: 7.4 =B1 1.3 y) children. The
diagnosis of autism was based on the criteria for autistic disorder as
defined in the Diagnostic and Statistical Manual of Mental Disorders,
Fourth Edition (DSM-IV) and by a diagnostic interview conducted by a
developmental pediatrician. Of the 20 autistic children, all were
white, 14 were boys, 6 were girls, 19 were diagnosed with regressive
autism, and 1 had infantile autism. Most of these children were
impaired in speech and socialization skills and exhibited symptoms of
gastrointestinal distress; before the study, 16 were taking a
multivitamin and mineral supplement containing 400 =B5g folic acid and 3
=B5g vitamin B-12. None of the autistic children were taking prescribed
medicines, such as valproic acid or anticonvulsants, that might have
affected methionine metabolism. A quantifiable diet questionnaire was
not administered as part of this study; thus, specific dietary
differences within and between groups cannot be determined. The control
subjects in the metabolic study were healthy white US children with no
history of chronic disease or autism who had participated in a similar
baseline study of children with Down syndrome (15). Control children
took over-the-counter vitamin supplements and were not taking
medications known to interfere with methionine metabolism. Exclusion
criteria for both groups included a diagnosis of malnutrition, the
presence of active infection, or known genetic disease.

The protocol and informed consent for this study were reviewed and
approved by the Institutional Review Board at the University of
Arkansas for Medical Sciences. The details of the study were explained
to the parents of the participating children, and written informed
consent was obtained from the parents.

Study design
The metabolic study consisted of 3 parts. In the first component,
baseline concentrations of plasma metabolites in the methionine cycle
and transsulfuration pathway were measured in 20 autistic children and
compared with plasma concentrations in 33 control children to establish
whether the metabolic profile of the autistic children differed
significantly from that of the control children. In the second
component, based on the observed abnormalities in plasma metabolites, a
subset of 8 autistic children were given oral supplements of 800 =B5g
folinic acid and 1000 mg betaine (anhydrous trimethylglycine) twice a
day in an attempt to improve the metabolic profile; this is referred to
as intervention 1. After 3 mo on this regimen, blood samples were again
taken and the metabolite concentrations were compared with baseline
concentrations of each metabolite. In the third component, the same
subset of 8 children were given an injectible form of methylcobalamin
(75 =B5g/kg) twice a week in addition to the oral folinic acid and
betaine for an additional month; this is referred to as intervention 2.
Each child served as his or her own control for the intervention study.

Nutritional supplements
USP-grade folinic acid was obtained from Douglas Laboratories
(Pittsburgh) or Thorne Research, Inc (Dover, ID) and was given twice a
day as 800 =B5g oral powder in juice. Betaine (trimethylglycine, USP
grade) was purchased from Life Extension Foundation (Fort Lauderdale,
FL) and given twice a day as 1000 =B5g oral powder in juice. USP
methylcobalamin was obtained from Hopewell Pharmaceuticals (Hopewell,
NJ) or Unique Pharmaceuticals (Temple, TX) as an injectible liquid and
given subcutaneously at a dose of 75 =B5g/kg twice a week.

Sample treatment and HPLC method
Fasting blood samples were collected into EDTA-containing evacuated
tubes (B-D Biosciences, Dallas) and immediately chilled on ice before
being centrifuged at 4000 x g for 10 min at 4 =B0C. Plasma aliquots were
transferred into cryostat tubes and stored at -80 =B0C until
extraction and HPLC quantification. For determination of methionine,
total homocysteine, cysteine, and total glutathione (tGSH)
concentrations, 50 =B5L of a freshly prepared solution of 1.43 mmol
sodium borohydride/L containing 1.5 =B5mol EDTA/L, 66 mmol NaOH/L, and
10 =B5L isoamyl alcohol was added to 200 =B5L plasma to reduce all
sulfhydryl bonds. The samples were incubated at 40 =B0C in a shaker for
30 min. To precipitate proteins, 250 =B5L ice-cold 10% metaphosphoric
acid was added and mixed well, and the sample was incubated for an
additional 10 min on ice. After centrifugation at 18 000 x g for 15 min
at 4 =B0C, the supernatant fluid was filtered through an 0.2-=B5m nylon
membrane filter (PGC Scientific, Frederic, MD), and a 20-=B5L aliquot
was injected into the HPLC system. For measurement of SAM, SAH,
adenosine, cystathionine, and oxidized glutathione (GSSG)
concentrations, 100 =B5L of 10% metaphosphoric acid was added to 200 =B5L
plasma to precipitate protein; the solution was mixed well and
incubated on ice for 30 min. After centrifugation for 15 min at 18 000
g at 4 =B0C, supernatant fluids were passed through an 0.2-=B5m nylon
membrane filter, and 20 =B5L was injected into the HPLC system.

The details of the method for HPLC elution and electrochemical
detection were described previously (18, 19). The separation of
metabolites was performed by using HPLC with a Shimadzu solvent
delivery system (ESA model 580) and a reverse-phase 5-=B5m C18 column
(4.6 x 150 mm; MCM Inc, Tokyo) obtained from ESA Inc (Chemsford, MA). A
20-=B5L aliquot of plasma extract was directly injected onto the column
by using a Beckman Autosampler (model 507E; Beckman Instruments,
Irvine, CA). All plasma metabolites were quantified by using model
5200A Coulochem II and CoulArray electrochemical detection systems
equipped with a dual analytic cell (model 5010), a 4-channel analytic
cell (model 6210), and a guard cell (model 5020) (all: ESA Inc). The
unknown concentrations of plasma metabolites were calculated from peak
areas and standard calibration curves with the use of HPLC software.

Statistical analysis
Metabolic data are presented as means =B1 SDs. Statistical differences
in plasma metabolites between case and control children were
ascertained by using the Student's t test with significance set at
0=2E05. One-way analysis of variance was performed to ascertain whether
differences existed between plasma metabolite concentrations at the 3
time points: baseline (no intervention), after intervention 1 (folinic
acid and betaine), and after intervention 2 (folinic acid, betaine, and
methylcobalamin). Individual metabolites at baseline were subsequently
compared with those after intervention 1 and intervention 2 by using
the paired Student's t test with the Bonferroni correction. Statistical
analyses were accomplished with the use of SIGMASTAT software (version
2=2E0; Systat Software Inc, Richmond. CA).

RESULTS

Baseline methionine cycle and transsulfuration pathway metabolites
The baseline concentrations of metabolites in the methionine cycle and
in the transsulfuration pathway were significantly different between
the autistic children and the control children. Within the methionine
cycle, plasma concentrations of methionine, SAM, and homocysteine were
significantly lower and SAH and adenosine concentrations were
significantly higher than those in the control children (Table 1). The
ratio of SAM to SAH was almost 50% lower in the autistic children than
in the control children. The significant reductions in plasma
cystathionine and cysteine concentrations observed in the autistic
children (Table 1) were consistent with a decrease in CBS-mediated
transsulfuration. Associated with the low mean plasma cysteine
concentration was a significant decrease in tGSH concentrations. GSSG
was increased almost twofold, and tGSH:GSSG was reduced by 70%.


TABLE 1 Comparison of methionine cycle and transsulfuration
metabolites between autistic children and control children1

Supplementation with folinic acid and betaine (intervention 1)
A subset of 8 autistic children participated in an intervention trial
designed to improve their metabolic profile. Oral supplementation with
800 =B5g folinic acid and 1000 mg betaine, both given twice a day, was
maintained for a period of 3 mo (intervention 1), and a second blood
sample was drawn. Relative to baseline concentrations, mean plasma
methionine, SAM, homocysteine, cystathionine, cysteine, and tGSH
concentrations and SAM:SAH and tGSH:GSSG in these 8 children were
higher (Table 2). In addition, the high SAH and adenosine
concentrations observed at baseline decreased with the betaine and
folinic acid supplements during intervention 1. The mean concentrations
of methionine, SAM, SAH, adenosine, and homocysteine were not
statistically different from those in the control children, which
indicated that intervention with folinic acid and betaine had brought
these methionine cycle metabolites into the normal range. Although
supplementation was effective in normalizing the methionine cycle
metabolites to the concentrations in the control subjects, the
intervention significantly improved but did not normalize tGSH or GSSG
concentrations or tGSH:GSSG.


Supplementation with folinic acid, betaine, and methyl vitamin B-12
(intervention 2)
For intervention 2, an injectible form of methylcobalamin (75 =B5g/kg)
was added to the folinic acid and betaine regimen for a period of 1 mo,
after which the third blood sample was taken for HPLC analysis. The
addition of injectible methylcobalamin (intervention 2) did not alter
the mean concentrations of methionine, SAM, SAH, or homocysteine beyond
the alterations induced by the intervention with folinic acid and
betaine (Table 2). However, relative to intervention 1, the addition of
injectible methylcobalamin further decreased the concentrations of
adenosine and GSSG and further increased the concentrations of
methionine, cysteine, and tGSH and SAM:SAH and tGSH:GSSG.


DISCUSSION

Autism is a complex neurodevelopmental disorder that is thought to
involve an interaction between multiple, variable susceptibility genes
(21), epigenetic effects (22), and environmental factors (23). The
apparent increase in the diagnosis of autistic-spectrum disorders from
4-5 in 10 000 children in the 1980s to 30-60 in 10 000 children in
the 1990s has raised great concern (24-27). This increased prevalence
of autism has enormous future public health implications and has
stimulated intense research into potential etiologic factors and
candidate genes. Because abnormal folate metabolism and low glutathione
concentrations have been reported in other neurologic disorders,
including Alzheimer disease, Parkinson disease, schizophrenia, and Down
syndrome (15, 28-31), we measured the concentrations of methionine
methylation and transsulfuration metabolites in a cohort of autistic
children.

The concentrations of metabolites among the control children in this
study were within the range of values previously found in several
studies (32-34). The observed imbalance in methionine and
homocysteine metabolism in the autistic children is complex and not
easily explained by perturbation of a single pathway or isolated
genetic or nutritional deficiency. Within the methionine cycle,
significant decreases in plasma concentrations of methionine, SAM, and
homocysteine were associated with significant increases in adenosine
and SAH. The low methionine and SAM concentrations would suggest a
reduction in methionine synthase activity; however, the observed
decrease in homocysteine does not fit that interpretation. The data may
be best explained by oxidative inactivation of methionine synthase in
combination with a decrease in SAH hydrolase activity secondary to the
increase in adenosine (35, 36). Adenosine binds to the active site of
SAH hydrolase, and increased concentrations of adenosine have been
shown to reduce SAHH activity (36, 37). A combined enzyme deficit would
also be consistent with the observed decrease in SAM and increase in
SAH concentrations. In this case, the decrease in homocysteine
concentrations would reflect an adenosine-mediated decrease in SAH
hydrolysis and homocysteine synthesis. The functional consequence of an
increase in SAH is product inhibition of most cellular
methyltransferases (38). Low methionine and SAM concentrations in
combination with increased SAH and adenosine concentrations were shown
previously to be associated with reduced cellular methylation capacity
(39). The twofold decrease in SAM:SAH also suggests an impaired
capacity in these autistic children for cellular methylation.

The metabolic pattern observed in the transsulfuration pathway may
provide a more cohesive explanation for the unusual imbalance in
methionine cycle metabolites. Low concentrations of cystathionine,
cysteine, and tGSH are consistent with reduced flux through the
transsulfuration pathway. Furthermore, the significant increase in GSSG
disulfide and the 67% decrease in tGSH:GSSG indicate chronic oxidative
stress. Within the methionine cycle, methionine synthase, betaine
homocysteine methyltransferase, and methionine adenosyltransferase are
all redox-sensitive enzymes that are down-regulated by oxidative stress
(40-42). A decrease in methionine- and SAM-regulated CBS activity
would increase the requirement for cysteine, effectively making it an
essential amino acid in these children. Because cysteine is the
rate-limiting amino acid for glutathione synthesis, its decrease is
consistent with low concentrations of glutathione (43, 44). The
remarkably consistent decrease in cysteine and glutathione
concentrations and tGSH:GSSG in the autistic children suggests an
increased vulnerability to oxidative stress.

The genetic or environmental factors (or both) that would initiate
oxidative stress and abnormal metabolic profiles in the autistic
children are not clear. It is possibly relevant that, in autistic
children, decreased activity of adenosine deaminase and increased
frequency of adenosine deaminase polymorphisms have been shown to be
associated with low adenosine deaminase activity (14, 45). The observed
increase in adenosine could be due to either an inhibition of adenosine
kinase or an increase in 5-nucleotidase, both of which have been shown
to occur with oxidative stress (46, 47). Elevated intracellular
adenosine has been shown to inhibit glutathione synthesis (48, 49).
Alternatively, a genetic predisposition to environmental agents or
conditions that promote oxidative stress could contribute to the
abnormal metabolic profile observed in the autistic children.

The targeted nutritional intervention trial in a subset of the autistic
children was specifically designed to increase methionine
concentrations (intervention 1). Betaine homocysteine methyltransferase
provides a folate-vitamin B-12-independent pathway in the liver and
kidney to remethylate homocysteine to methionine (17). Supplemental
betaine (trimethylglycine) has been shown to up-regulate betaine
homocysteine methyltransferase expression and activity to increase
methionine synthesis (50). Folinic acid (5-formyl tetrahydrofolate) was
used rather than folic acid because the former is absorbed as the
reduced metabolite and can enter folate metabolism as 5,10-methylene
tetrahydrofolate, thereby reducing the possibility of promoting a
folate trap (51, 52). As shown in Table 2, the intervention with
betaine and folinic acid was successful in bringing all the metabolites
within the methionine cycle into the normal range and simultaneously
improving significantly the metabolites in the transsulfuration
pathway. The increase in methionine, SAM, and homocysteine
concentrations and the decrease in adenosine and SAH concentrations
suggested that the intervention stimulated an increased flux through
the methionine cycle. In addition, the significant increase in
cystathionine concentrations suggests that the supplements were
effective also in increasing CBS activity, most likely because of
up-regulation by the increase in SAM. The associated increases in
cysteine and glutathione indicate that transsulfuration to glutathione
was enhanced by the supplements. The decrease in adenosine is
consistent with a concomitant release of SAHH inhibition and decrease
in SAH and, possibly, the release of a bottleneck in methionine cycle
turnover. The mechanism for the decrease in adenosine concentrations,
however, is not clear. One possibility is that the increase in cysteine
or glutathione concentration (or both) relieved the need for adenosine
as a protective factor against oxidative damage (53, 54).

The addition of injectible methylcobalamin to the protocol
(intervention 2) was based on empirical observations of clinical
improvement in speech and cognition (by JAN) and the possibility that
it might enhance methionine synthase activity under conditions of
oxidative stress by replacing oxidized inactive coenzyme B-12
[cob(II)alamin] or by posttranslational up-regulation of methionine
synthase, or both (55, 56). One month after the addition of
methylcobalamin, the methionine concentrations were within the control
range (Table 1), and further improvements in adenosine and SAH
concentrations and SAM:SAH were observed. Unexpectedly, and perhaps
most significantly, the addition of methylcobalamin reduced the
concentrations of inactive GSSG and increased the tGSH concentrations
and tGSH:GSSG so that they were not different from those in the control
children (Table 1). These positive changes in the glutathione redox
profile most likely reflect the increase in cysteine as the
rate-limiting amino acid for glutathione synthesis (44). Of note, there
is a higher demand for cysteine (and, indirectly, methionine) for de
novo glutathione synthesis during chronic oxidative stress (43). Low
antioxidant enzyme activity in autistic children has been reported in 3
recent studies (57-59) that provide additional support for oxidative
stress as a part of the etiology of autism. If the decreases in plasma
methionine, cysteine, and glutathione concentrations in autistic
children observed in the current study are confirmed in a larger study,
low concentrations of these thiol metabolites could provide metabolic
biomarkers for autism.

Although clinical improvements in speech and cognition were noted by
the attending physician (PC), they were not measured in a quantifiable
manner and are therefore not reported here. Specific dietary
differences between groups could have contributed to our results, but
we consider it unlikely that uniform dietary differences within the
autistic group existed that could have accounted for the remarkably
consistent metabolic alterations. Increased frequency of common
polymorphisms in these pathways may have contributed to the observed
metabolic phenotype, and studies of that subject, as well as studies to
quantify clinical improvement, are currently underway. Our attempts to
interpret these preliminary metabolic findings are clearly speculative,
and a better understanding of the abnormal one-carbon metabolism in
these children will require additional research efforts. Nonetheless,
the ability to correct the metabolic imbalance with targeted
nutritional intervention implies that certain aspects of autism may be
treatable.

Nineteen of the 20 children participating in the study were diagnosed
with "regressive" autism (apparently normal development until
regression into autism between ages 1.5 and 3 y). On the basis of their
abnormal metabolic profiles, we hypothesize that an increased
vulnerability to oxidative stress (environmental, intracellular, or
both) and impaired methylation capacity may contribute to the
development and clinical manifestation of regressive autism.


ACKNOWLEDGMENTS

SJJ was responsible for study design, study coordination,
interpretation of data, and manuscript writing. PC was responsible for
patient recruitment, obtaining supplements, patient compliance,
monitoring clinical status, and methylcobalamin injections. SM was
responsible for HPLC quantification of plasma metabolites and data
collection and interpretation. SJ was responsible for plasma and DNA
extraction and data collection and interpretation. LJ was responsible
for patient recruitment, study coordinating and consulting, and data
interpretation. DWG was responsible for statistical analysis of data.
JAN was responsible for initiating the methylcobalamin treatment in
autistic patients, providing consultation, and interpreting data. None
of the authors has any financial conflict of interest.


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Received for publication June 1, 2004. Accepted for publication August
23, 2004.

On Wed, 15 Dec 2004 08:45:28 GMT, "CWatters"
wrote:


"Jeff" wrote in message
...
Could you please show us the peer-reviewed research papers?

"Dr. Jill James reports in a peer-reviewed study published this month in the
American Journal of Clinical Nutrition"


LOL ... Jeffie uses that mantra whenever things go against the Quack
Vac Flacks ... we can only be joyful that he was never allowed to
"practice" with an unrestricted medical license ... and is lying when
he calls himself a Kids Doc ...

He was never certified as a Pediatrician ... he and Barrett just
pretend he was:

As far as the last line ... it's only been 2 years since they FINALLY
removed Mercury ...

Posted on Tue, Dec. 14, 2004


http://www.kentucky.com/mld/heraldle...n/10411444.htm


Study suggests mercury-autism link

BLOOD TESTS SHOW LOWER LEVELS OF DETOXIFYING ENZYME

By Tina Hesman

ST. LOUIS POST-DISPATCH


ST. LOUIS - Children with autism might process mercury differently
than most children, leaving them susceptible to damage from
preservatives in vaccines and other sources of the heavy metal,
according to a controversial report released yesterday.

The report, by the independent Environmental Working Group, highlights
the research of S. Jill James, a professor of biochemistry and
pediatrics at the University of Arkansas for Medical Sciences.

James conducted blood tests on 20 children with autism and compared
them with blood samples from 33 children who do not have autism. She
found lower levels of a mercury-detoxifying chemical, glutathione, in
the blood of autistic children.

The enzyme also helps rid the body of other heavy metals that might
damage cells and organs.

James also found that supplementing the autistic children's diets with
a combination of folinic acid, betaine and methyl vitamin B-12 brought
glutathione levels back to levels seen in the children in the control
group.

The studies again raise concerns that some genetically susceptible
children are more prone to neurological damage when exposed to mercury
and other toxic substances in the environment. Vaccines, fish and
dental amalgam fillings have been fingered as sources of mercury.

The vaccine connection to the disorder has been the subject of intense
debate. Parents and some scientists say the use of a mercury-based
preservative called thimerosal in vaccines corresponded with an
explosion in the number of autism cases. Thimerosal was taken out of
vaccines in 2002, but the rate of autism remains at the same level as
before it was removed.

OK, now that I'm finished chasing ghosts...

The paper is not in the "American Journal of Nutrition Research", but
instead is in "Neurotoxicology" (Jan. 26:1-8.2005). It uses two cancer
cell lines, a neuroblastoma (ATCC #CRL-2266, trisomy 1, 47 chromosomes)
and a glioblastoma (ATCC #CRL-2020, 87 to 91 chromosomes, depending on
your subculture), to draw its conclusions.

Here's one sentence from the paper:

"Acute high dose exposures to Thimerosal (=B5mol/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)."

So, it's high dose. In other words, it's probably not biologically
relevant. If I double the molar concentration of sodium chloride (table
salt) in my cell cultures, the cells die in a matter of minutes.

Then there's this at the end:

"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."

Wow, talk about grand statements. I'm surprised the reviewers let this
one get by. To go from a cell culture experiment to the whole body is
quite a leap.

"Ilena Rose" wrote in message
...
On Wed, 15 Dec 2004 08:45:28 GMT, "CWatters"
wrote:


"Jeff" wrote in message
...
Could you please show us the peer-reviewed research papers?

"Dr. Jill James reports in a peer-reviewed study published this month in
the
American Journal of Clinical Nutrition"


LOL ... Jeffie uses that mantra whenever things go against the Quack
Vac Flacks ... we can only be joyful that he was never allowed to
"practice" with an unrestricted medical license ... and is lying when
he calls himself a Kids Doc ...

Nice personal attack. I just asked for the studies. If wanting people to
back up their statements makes me a Quack Vac Flack, then I am proud to be
one called one.

He was never certified as a Pediatrician ... he and Barrett just
pretend he was:

I never pretended I was "certified as a Pediatrician."

garbage deleted

Jeff