S Jill Article on Metabolic Biomarkers - Full Text

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American Journal of Clinical Nutrition, Vol. 80, No. 6, 1611-1617, December

2004

© 2004 American Society for Clinical Nutrition

________________________________________

ORIGINAL RESEARCH COMMUNICATION

Metabolic biomarkers of increased oxidative stress and impaired methylation

capacity in children with autism1,2

S Jill , Cutler, Stepan Melnyk, Stefanie Jernigan, Laurette Janak,

W Gaylor and A Neubrander

1 From the Department of Pediatrics, University of Arkansas for Medical

Sciences, and the Arkansas Children's Hospital Research Institute, Little

Rock, AR (SJJ, SM, and SJ); Niagara Falls, NY (PC); Colden, NY (LJ); Gaylor

and Associates, LLC, Eureka Springs, AR (DWG); and Edison, NJ (JAN)

2 Reprints not available. Address correspondence to SJ , Arkansas

Children's Hospital Research Institute, Slot 512-40B, 1120 Marshall Street,

Little Rock, AR 72202. E-mail: jamesjill@....

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 • biomarkers • oxidative stress • methylation •

methionine • S-adenosylmethionine • S-adenosylhomocysteine • adenosine •

cysteine • 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 ß-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.

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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 ß-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 ( ± SD age: 6.4 ±

1.5 y) and 33 control (age: 7.4 ± 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 µg folic acid and 3 µg

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 µg 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 µg/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 Laboratories (Pittsburgh)

or Thorne Research, Inc (Dover, ID) and was given twice a day as 800 µg oral

powder in juice. Betaine (trimethylglycine, USP grade) was purchased from

Life Extension Foundation (Fort Lauderdale, FL) and given twice a day as

1000 µg 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 µg/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 °C. Plasma aliquots were transferred

into cryostat tubes and stored at –80 °C until extraction and HPLC

quantification. For determination of methionine, total homocysteine,

cysteine, and total glutathione (tGSH) concentrations, 50 µL of a freshly

prepared solution of 1.43 mmol sodium borohydride/L containing 1.5 µmol

EDTA/L, 66 mmol NaOH/L, and 10 µL isoamyl alcohol was added to 200 µL plasma

to reduce all sulfhydryl bonds. The samples were incubated at 40 °C in a

shaker for 30 min. To precipitate proteins, 250 µL 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 °C, the supernatant fluid was filtered through an 0.2-µm nylon

membrane filter (PGC Scientific, Frederic, MD), and a 20-µL aliquot was

injected into the HPLC system. For measurement of SAM, SAH, adenosine,

cystathionine, and oxidized glutathione (GSSG) concentrations, 100 µL of 10%

metaphosphoric acid was added to 200 µL 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 °C, supernatant fluids were

passed through an 0.2-µm nylon membrane filter, and 20 µL 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-µm C18 column (4.6 x 150 mm; MCM Inc, Tokyo)

obtained from ESA Inc (Chemsford, MA). A 20-µL 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 ± SDs. Statistical differences in

plasma metabolites between case and control children were ascertained by

using the Student's t test with significance set at 0.05. 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 2.0; 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%.

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TABLE 1 Comparison of methionine cycle and transsulfuration metabolites

between autistic children and control children1

Control children (n = 33) Autistic children (n = 20)

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Methionine (µmol/L) 31.5 ± 5.7 (23–48) 19.3 ± 9.7 (15–25)2

SAM (nmol/L) 96.9 ± 12 (77–127) 75.8 ± 16.2 (68–100)3

SAH (nmol/L) 19.4 ± 3.4 (16–27) 28.9 ± 7.2 (14–41)2

SAM:SAH 5.2 ± 1.3 (4–8) 2.9 ± 0.8 (2–4)2

Adenosine (µmol/L) 0.27 ± 0.1 (0.1–0.4) 0.39 ± 0.2 (0.17–0.83)4

Homocysteine (µmol/L) 6.4 ± 1.3 (4.3–9.0) 5.8 ± 1.0 (4.0–5.8)3

Cystathionine (µmol/L) 0.17 ± 0.05 (0.1–0.27) 0.14 ± 0.06 (0.04–0.2)5

Cysteine (µmol/L) 202 ± 17 (172–252) 163 ± 15 (133–189)2

tGSH (µmol/L) 7.6 ± 1.4 (3.8–9.2) 4.1 ± 0.5 (3.3–5.2)2

Oxidized glutathione (nmol/L) 0.32 ± 0.1 (0.11–0.43) 0.55 ± 0.2

(0.29–0.97)2

tGSH:GSSG 25.5 ± 8.9 (13–49) 8.6 ± 3.5 (4–11)2

1 All values are ± SD; range in parentheses. SAM, S-adenosylmethionine;

SAH, S-adenosylhomocysteine; tGSH, total glutathione; GSSG, oxidized

glutathione.

2 Significantly different from control children: P < 0.001,

3 Significantly different from control children: P < 0.01,

4 Significantly different from control children: P < 0.05,

5 Significantly different from control children: P < 0.002.

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

µg 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.

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TABLE 2 Results of intervention trials1

Baseline (n = 8) Intervention 1 (n = 8) P2 Intervention

2 (n = 8) P3

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Methionine (µmol/L) 19.2 ± 3.5 25.7 ± 3.6 0.04 30.9 ± 7.7

NS

SAM (nmol/L) 75.5 ± 5.0 112.9 ± 20.8 0.008 101.6 ± 20.5 NS

SAH (nmol/L) 27.6 ± 6.1 16.9 ± 6.5 0.002 14.3 ± 7.5 NS

SAM:SAH 2.9 ± 0.8 7.4 ± 4.1 0.004 8.9 ± 4.5 0.04

Adenosine (µmol/L) 0.30 ± 0.2 0.18 ± 0.04 0.08 0.14 ± 0.03

0.002

Homocysteine (µmol/L) 5.4 ± 0.9 6.7 ± 0.7 0.05 7.4 ± 1.7

NS

Cystathionine 0.10 ± 0.02 0.22 ± 0.08 0.01 0.25 ± 0.08 NS

Cysteine (µmol/L) 166 ± 11.4 180 ± 11 0.02 199.3 ± 15

0.002

tGSH (µmol/L) 4.0 ± 0.7 5.0 ± 0.9 0.002 6.7 ± 1.6

0.016

Oxidized glutathione (nmol/L) 0.59 ± 0.2 0.38 ± 0.1 0.08 0.25

± 0.05 0.008

tGSH:GSSG 7.5 ± 2.3 13.8 ± 3.9 0.008 28.7 ± 7.1

0.002

1 All values are ± SD. SAM, S-adenosylmethionine; SAH,

S-adenosylhomocysteine; tGSH, total glutathione; GSSG, oxidized glutathione.

Intervention 1: 800 µg folinic acid and 1000 mg betaine were administered

twice a day from immediately after the baseline blood draw for a period of 3

mo; intervention 2: subcutaneous injection of 75 µg methylcobalamin/kg was

added to folinic acid and betaine supplementation for an additional month.

2 Intervention 1 compared with baseline.

3 Intervention 2 compared with intervention 1.

Supplementation with folinic acid, betaine, and methyl vitamin B-12

(intervention 2)

For intervention 2, an injectible form of methylcobalamin (75 µg/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.