Autism Research in Arkansas:

On-going clinical trials

and the Arkansas Autism Alliance

S. Jill James, PhD

Professor, Department of Pediatrics
Director, Autism Metabolic Genomics Laboratory
Arkansas Children’s Hospital Research Institute
University of Arkansas for Medical Sciences
Little Rock, AR
 OVERVIEW
Review of metabolic pathways: folate/methionine/glutathione

Efficacy of methylB12 and folinic acid treatment on

glutathione redox status and core behaviors in autism

Parent Metabolic Profiles

Specific Aims of our 5 year NIH-funded study

Placebo-controlled double-blind cross-over study of

broad spectrum nutritional supplementation

AAA and ATN in Arkansas

 Methionine Transsulfuration to Cysteine and Glutathione
Methionine

THF

5,10-CH2THF 1 MS
B12
MTHFR
5-CH3THF

Homocysteine

B6

THF: tetrahydrofolate
Enzymes
 Methionine Transsulfuration to Cysteine and Glutathione
Methionine
Methylation
THF SAM
Potential
(SAM/SAH)
MTase
5,10-CH2THF 1 MS 2 Cell Methylation
B12 SAH
MTHFR
5-CH3THF SAHH
Adenosine
Homocysteine

B6

THF: tetrahydrofolate
Enzymes
 Methionine Transsulfuration to Cysteine and Glutathione
Methionine
Methylation
THF SAM
Potential
(SAM/SAH)
MTase
5,10-CH2THF 1 MS 2 Cell Methylation
B12 SAH
MTHFR
5-CH3THF SAHH
Adenosine
Homocysteine

B6
B6 CBS
Cystathionine
B6 Antioxidant
3
THF: tetrahydrofolate Cysteine Redox Potential
Enzymes (GSH/GSSG)
GSH GSSG
Methionine Transsulfuration to Cysteine and Glutathione
Methionine
Methylation
THF SAM
Potential
(SAM/SAH)
MTase
5,10-CH2THF 1 MS 2 Cell Methylation
B12 SAH
MTHFR
5-CH3THF SAHH Adenosine

Homocysteine
1 Folate Cycle CBS
B6
B6
Cystathionine
2 Methionine Cycle B6 Antioxidant
3
Cysteine Redox Potential
3 Transsulfuration (GSH/GSSG)
Pathway GSH GSSG
 Vital Importance of these Interdependent Metabolic Pathways

Methionine

METHYLATION
THF SAM
MTase Cellular
5,10-CH2THF 1 MS 2 Methylation
B12 SAH Reactions

5-CH3THF SAHH
Purines and Adenosine
Thymidylate
Homocysteine

DNA SYNTHESIS Cystathionine

B6
REDOX
3 Cysteine HOMEOSTASIS
PROLIFERATION
GSH GSSG
AN OPEN LABEL TRIAL OF METHYLCOBALAMIN
AND FOLINIC ACID IN AUTISTIC CHILDREN
Can supplementation with methyl-B12 and folinic Acid
improve glutathione levels and core behaviors
in autistic children?

Intervention: MethylB12 (75µg/Kg every 3 days)

(3 months) Folinic Acid (400 µg bid)

Inclusion Criteria: Autistic Disorder (DSM-IV; CARS)

Age 3-7
No previous supplements
GSH < 6.0

Endpoints: Methylation and glutathione metabolites

Vineland Adaptive Behavioral Scales
 STUDY DESIGN

Each child served as their own control in the open label trial in
which both parents and investigators were aware that the child
was receiving supplements ofmethyl-B12 and folinic acid for a
period of three months.

Plasma metabolites in the transmethylation and transsulfuration

pathways were measured at baseline and again after the 3 month
intervention period.

The study nurse administered and scored the Vineland Adaptive

Behavior Scales parent questionnaire before and after the 3
month intervention.
 Methyl B12

Folinic Acid Methionine

THF SAM
MTase Cellular
5,10-CH2THF 1 MS 2 Methylation
B12 SAH Reactions

5-CH3THF SAHH
Purines and Adenosine
Thymidylate
Homocysteine
Folinic Acid
DNA SYNTHESIS Cystathionine
B6

3 Cysteine

GSH GSSG
 METABOLIC DATA
Plasma Metabolite Control Autism Autism
Concentration Children Pre-treatmentb Post-treatment p valuea
(n = 42) (n = 40) (n = 40)

Methionine 24 ± 3 21 ± 4 22 ± 3 ns
SAM (nmol/L) 78 ± 22 66 ± 13 69 ± 12 ns
SAH (nmol/L) 14.3 ± 4.3 15.2 ± 5 14.8 ± 4 ns
SAM/SAH (µmol/L) 5.6 ± 2.0 4.7 ± 1.5 5.0 ± 2.0 ns
Homocysteine (µmol/L) 5.0 ± 1.2 4.8 ± 1.8 5.3 ± 1.1 0.04
Cysteine (µmol/L) 210 ± 18 191 ± 24 215 ± 19 0.001
Total Glutathione (µmol/L) 7.5 ± 1.8 5.4 ± 1.3 6.2 ± 1.2 0.001
Free Glutathione (µmol/L) 2.8 ± 0.8 1.5 ± 0.4 1.8 ± 0.4 0.008
GSSG (µmol/L) 0.18 ± 0.07 0.28 ± 0.08 0.22 ± 0.06 0.001
tGSH/GSSG 47 ± 18 21 ± 6 30 ± 9 0.001
fGSH/GSSG 17 ± 6.8 6±2 9±3 0.001
a
P value refers to treatment effect
 Cysteine
300

250
µmol/L

200

150

100
Before After

50

0
 Total Glutathione
10
9 x

8
7
6
µmol/L

5
4
3
Before After
2
1
0
 GSSG

0.6

0.5

0.4
µmol/L

0.3

0.2

0.1
Before After
0
 Total GSH/GSSG
60

50

40

30

20

10

Before After
0
 SUMMARY OF METABOLIC RESULTS

1. All baseline metabolites were significantly different

from age-matched controls (except for SAH)
2. The treatment did not significantly improve
levels of methionine, SAM or SAM/SAH
• The treatment did significantly improve cysteine,
glutathione, and GSH/GSSG
10. Although significantly improved, glutathione and
GSH/GSSG did not reach levels in control children
 Behavioral Evaluation

The Vineland Adaptive Behavior Scales (VABS)

provides a numerical score for adaptive functioning in
the areas of communication, socialization, daily living
skills, motor skills, and an adaptive behavior
composite (ABC) score.

The data are presented as the mean score for each

category before and after intervention.
 BEHAVIOR SCORES
Vineland Category Baseline Post-Treatment
Score Score Change in Score p value
(mean ± SD) (mean ± SD) (mean; 95% C I)

Communication 65.3 ± 12.9 72.0 ± 15.5 6.7 (3.5, 10) <0.001

Daily Living Skills 67.0 ± 76 76.0 ± 17.7 9.0 (4.0, 14) <0.007

Socialization 68.2 ± 9.3 75.7 ± 14.7 7.5 (3.5, 11) <0.005

Motor Skills 75.6 ± 9.7 79.0 ± 14.7 3.3 (0, 8) 0.12

Composite Score 66.5 ± 9.2 73.9 ± 17.0 6.6 (2.3, 11) <0.003
 SUMMARY OF BEHAVIOR RESULTS
Although treatment with methylB12 and folinic acid
significantly improved core behaviors, they did not reach
standard scores for unaffected children (100 ± 15)

CONCLUSIONS
Improvement in measures of both metabolic and behavioral
endpoints converge to suggest that some children may
benefit from targeted nutritional intervention
What about the parents?
 Maternal Methionine Cycle Metabolites:
Autism Moms Control Moms
(n = 46) (n= 200)

Methionine (µM/L) 24 ± 5 26 ± 6

SAM (nM/L) 80 ± 19 83 ± 13

SAH (nM/L) 33 ± 14* 23 ± 8.4

SAM/SAH Ratio 3.1 ± 1.7* 4.0 ± 1.4

Homocysteine (µM/L) 11 ± 3.9* 7.6 ± 1.6

*statistically significant

It would be a very good idea to ask your physician to check

your “total” homocysteine
 Maternal Transsulfuration Metabolites

Autism Moms Control Moms

Cysteine (µM/L) 232 ± 40 231 ± 20

Total GSH (µM/L) 5.1 ± 1.7* 7.3 ± 1.5

Free GSH (µM/L) 1.5 ± 0.5* 2.6 ± 0.6

GSSG (µM/L) 0.30 ± 0.08* 0.24 ± 0.04

Total GSH/GSSG 17 ± 8 31 ± 10*

*statistically significant
 Metabolite imbalance and the risk of
being a mother of a child with autism

Control Case
Stratified Group Mothers Mothers Odds Ratio
(N=200) (N=46) (Risk)
SAH >30µMol/L) 14% 54% 6.9
SAM/SAH <2.5 10% 54% 10.7
tGSH/GSSG <20 11% 65% 15.2
SAM/SAH <2.5 and 3% 41% 46
tGSH/GSSG <20
 IMPORTANT CAVEAT

It is not possible to determine from this data

whether the abnormal metabolic profile in parents is
genetically determined or whether it simply reflects
the stress of living with an autistic child
 METABOLIC BIOMARKERS OF AUTISM:
PREDICTIVE POTENTIAL AND GENETIC SUSCEPTIBILITY

A 5 YEAR NIH-FUNDED STUDY (2006-2011)

SPECIFIC AIM 1: METABOLITES AND BEHAVIOR

Specific Aim 1: To determine whether the observed metabolite

imbalance is associated with quantitative measures of autistic
behavior
An expanded database of metabolic profiles will allow us to
determine whether the severity and specificity of the metabolite
imbalance is associated with the severity and specificity of behavioral
abnormalities.
 SPECIFIC AIM 2: PROSPECTIVE STUDY

Specific Aim 2: To investigate whether the abnormal metabolic

profile precedes the diagnosis of autism among toddlers 18-30
months of age who are identified in developmental delay clinics
to be at increased risk of developing autism.
 SPECIFIC AIM 2: PROSPECTIVE STUDY

M-CHAT autism screening test and plasma metabolic

biomarkers will be measured at Visit 1 and children will
be followed for subsequent diagnosis of autism (case) or
developmental delay (control).

Metabolic data will be analyzed statistically to determine

whether metabolic abnormalities precede the behavioral
diagnosis of autism and could serve as predictive
biomarkers for risk of autism.
AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk PASS = Developmental Delay and Normal

CONTROLS

Metabolic Profile Metabolic Profile

 AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk PASS = Developmental Delay and Normal

CONTROLS

Metabolic Profile Metabolic Profile

(1-6 months) (6 months)
Visit 2: M-CHAT Repeat M-CHAT Repeat
 AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk PASS = Developmental Delay and Normal

CONTROLS

Metabolic Profile Metabolic Profile

(1-6 months) (6 months)
Visit 2: M-CHAT Repeat M-CHAT Repeat

FAIL PASS

Visit 3: DSM-IV; CARS; ADOS Not Autism

Autism Diagnosis Control

 AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk PASS = Developmental Delay and Normal

CONTROLS

Metabolic Profile Metabolic Profile

(1-6 months) (6 months)
Visit 2: M-CHAT Repeat M-CHAT Repeat

FAIL FAIL = High risk Regression PASS

Visit 3: DSM-IV; CARS; ADOS Not Autism

Autism Diagnosis Control

 AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk PASS = Developmental Delay and Normal

CONTROLS
Baseline
Metabolic Profile Metabolic Profile
(1-6 months) (6 months)
Visit 2: M-CHAT Repeat M-CHAT Repeat

FAIL FAIL = High risk Regression PASS

Visit 3: DSM-IV; CARS; ADOS Not Autism

Final diagnosis
Autism Diagnosis Control
 IMPLICATIONS OF AIM 2
AUTISM PROSPECTIVE STUDY

If the metabolic profile is found to precede the

behavioral diagnosis, subsequent studies would
determine whether early intervention to
normalize the metabolic profile can reduce or
prevent the development of autism.
SPECIFIC AIM 3: CELLULAR CONSEQUENCES

Specific Aim 3: To establish whether cells from children

with autism exhibit evidence of increased oxidative stress
and oxidative damage.

This mechanistic aim will determine whether lymphocytes from

autistic children are inherently more vulnerable to oxidative
stress than control cells
 EXPERIMENTAL PROCEDURES

Lymphoblastoid cell lines from autistic children with

at least one affected sibling were compared with
unaffected control lymphoblastoid cell lines*

Pairs of autistic and control cells lines were cultured

under identical conditions. Rate of free radical
generation, GSH/GSSG were measured at baseline and
after exposure to thimerosal as oxidative stress.

*Preliminary data supported by SafeMinds

 Relative Free Radical Generation (DCF)

900 Control
800 Autistic
700
Vmax ROS Rate

600
500
400
300
200
100
0
0 0.3125 0.625 1.25 2.5
Thimerosal Concentration (uMol/L)
Cells from autistic children generate more free radicals than control cells
 Glutathione Redox Ratio (GSH/GSSG)
160
140 Control
120 Autistic
100
80
60
40
20
0
0 0.16 0.32 0.62 1.25 2.5
Thimerosal Concentration (uMol/L)

Cells from autistic children have lower GSH/GSSG ratio than control cells
 MITOCHONDRIAL REDOX IMBALANCE IN
LYMPHOBLASTOID CELL LINES

4 18
3.5 Autistic 16
GSH/GSSG RATIO
3 Control 14
12
2.5
10
2
8
1.5
6
1 4
0.5 2
0 0

fGSH GSSG (X 10) Control Autistic

 CONCLUSION

Since both cell lines were cultured at the same

time under identical conditions with identical media,
the differences at baseline and after exposure to
oxidant stress must reflect inherent genetic or
epigenetic differences.

These results provide experimental evidence that

cells from autistic children may be more sensitive
to pro-oxidant environmental exposures.
 SPECIFIC AIM 4: METABOLIC GENETICS
Specific Aim 4: Using a case-control design, we will
determine whether the frequency of relevant genetic
polymorphisms is increased among autistic children
and whether specific genotypes are associated with
the abnormal metabolic phenotype.

We have access to 500 trios (child, mother, father) from

NIH genetic repository to look at relevant SNP frequencies
and transmission
 A Targeted Approach to Autism Genetics:
Using the Metabolic Endophenotype as a
Guide to Candidate Genes
Methionine

THF
SAM Methyl Acceptor
DMG
5,10-CH2-THF B12 TC II Methyltransferase COMT

MTHFR Methylated Product

5-CH3-THF SAH
RFC
Homocysteine Adenosine

Cystathionine
CBS
Cysteine
GCL
Glutathione GST
Treating Oxidative Stress and the
Metabolic Pathology of Autism

A RANDOMIZED DOUBLE-BLIND PLACEBO-

CONTROLLED CROSS-OVER STUDY
 HYPOTHESIS

A significant proportion of autistic children have

impaired methylation and antioxidant/detoxification
capacity that results in chronic oxidative stress.

Targeted nutritional intervention that is designed to

correct the metabolic imbalance will significantly
improve their metabolic profile and improve measures
of autistic behavior.
 SPECIFIC AIMS

Specific Aim 1. We will screen children with a diagnosis of

autism for evidence of impaired methylation (↓SAM/SAH)
and impaired antioxidant capacity (↓GSH/GSSG)

Specific Aim 2. Children who exhibit evidence of impaired

methylation and antioxidant capacity will be randomized into
a double blind placebo-controlled cross-over trial of targeted
nutritional intervention designed to correct metabolic
deficiencies and to improve scores on standardized behavioral
evaluation tests.
 RANDOMIZED DOUBLE-BLIND PLACEBO-
CONTROLLED CROSS-OVER DESIGN
A is supplement first, placebo second
B is placebo first, supplement second

WASHOUT
A B
B A

Thiols, Complete Lab, Thiols, Complete Lab, Thiols, Complete Lab,

Behavioral Testing Behavioral Testing Behavioral Testing

Children are randomly assigned to either the placebo first or the treatment first
for 3 months before 1 month wash out period and cross-over
The supplements have been selected to impact
three core cellular functions that are altered with
chronic oxidative stress (www.clinicaltrials.gov)

1) Decreased SAM/SAH ratio and cellular

methylation capacity

2) Antioxidant and detoxification support

(mitochondrial and cytosolic)

3) Cell membrane integrity

 OUTCOME MEASURES
• Behavioral testing: ADOS; Vineland; PLS-2; SRS
Behavioral testing will be videotaped and administered by PhD
psychologists

3. Metabolic evaluation:
Plasma: Thiol profile; CBC; amino acid profile, P5P, HoloTCII;
sulfate; nitrotyrosine; lactate/pyruvate; 25-hydroxy
vitamin D; uric acid;
Urine: Sulfate, organic acids; creatinine; FIGlu, MMA
Cellular: RBC membrane phospholipids; leukocyte GSH/GSSG.

• Immunologic evaluation:
Flow cytometry for CRP, cytokine mRNA expression and protein
levels for TNFα; g-IFN, IL-1; IL-4, IL-6; IL-10; IL-13; T-regs
AUTISM TREATMENT NETWORK
(ATN) IN ARKANSAS
 The ATN
The ATN is a consortium of 15 national sites
composed of experts in developmental
pediatrics, neurology, genetics, metabolism,
sleep, and gastroenterology who are dedicated
to improving the standard of care of children
with autism.

The ATN believes that treatment of medical

issues can improve core behaviors and improve
quality of life for children and adults with autism
and their parents.
Our Dream for Autism in Arkansas

UAMS/ACH/ACHRI

Arkansas Autism Alliance (AAA)

 Clinical Evaluation & Treatment Center

UAMS/ACH/ACHRI
Arkansas
Autism Alliance

Resource and Outreach Center Translational Research Center

 FROM EPIDEMIOLOGY TO MECHANISM

BEHAVIOR

Necessary but Necessary but

Not Sufficient Not Sufficient

GENE EXPRESSION ENVIRONMENT

(Genetic/Epigenetic) (Vulnerability/Resistance)

Multiple, Additive Multiple, Additive

Variable Genes Variable Factors
 FROM EPIDEMIOLOGY TO MECHANISM

BEHAVIOR

Necessary but Necessary but

Not Sufficient Mechanism Not Sufficient
(Redox Imbalance;
Methylation)

GENE EXPRESSION Metabolic Endophenotype ENVIRONMENT

(GSH/GSSG) (SAM/SAH) (Vulnerability/Resistance)
(Genetic/Epigenetic)

Multiple, Additive Multiple, Additive

Variable Genes TREATMENT Variable Factors
 Acknowledgements
Autism Metabolic Genomics Laboratory
Stepan Melnyk, PhD
Stefanie Jernigan
Alena Savenka
Shannon Palmer
Sarah Blossom, PhD
Lesya Pavliv

Study Nurses
Nancy Chambers, Dana Schmidt,
Amanda Hubanks, Nancy Lowery