Microsoft Word - Needham et al Metabolomics Manuscript_Final.docx

Plasma and Fecal Metabolite

Profiles

Autism Spectrum

Disorder

Brittany D.

Needham

Mark D.

Adame

Gloria

Serena

Destanie

Rose

Gregory M.

Preston

Mary C.

Conrad

Stewart

Campbell

David H.

Donabedian

Alessio

asano

Paul

Ashwood

and Sarkis K.

Mazmanian

1

Biology and Biological Engineering, California Institute of Technology, Pasadena

91125, USA

2

Division of Pediatric Gastroenterology and Nutrition,

Mucosal Immunology and Biology Research

Center

, Massachusetts General Hospital for Children, Boston, MA, 02114, USA

3

Department of Medical Microbiology and Immunology,

University of California Davis

, Da

vis, CA,

95616, USA

4

The M.I.N.D. Institute, University of California, Davis, Sacramento, CA, 95817, USA

5

Axial Biotherapeutics

Waltham

, MA, 02

451

, USA

Correspondence:

bneedham@caltech.edu

and

sarkis@caltech.edu

Running title:

Plasma

and Fecal Metabolite Profiles in ASD

Keywords:

Autism Spectrum Disorder, ASD, metabolomics,

plasma metabolites, fecal metabolites,

steroid hormones, mitochondrial dysfunction,

phenolic metabolites

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ABSTRACT

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition with hallmark behavior

manifestations

including

impaired

social communication

and

restricted

repetitive behavior.

addition,

many

affected

individuals display

metabolic imbalances, immune dysregulation

gastrointestinal (GI) dysfunction

, and altered gut microbiome compositions

. We sought to

better

understand non

behavioral

features

of ASD by

determining

molecular

signatures

peripheral

tiss

ues

. Herein, we present

the

untargeted

metabolom

of 231 plasma and 97

fecal

samples from

large

cohort of

children with ASD and

typically developing

(

)

controls.

ifferences in lipid,

amino acid, and xenobiotic metabolism

discriminate

ASD and

sample

reveal

correlations between

specific

metabolite profiles and clinical behavior score

and

identify

metabolites

particularly

associated with

dysfunction in ASD.

These findings

support a

connection between GI

physiology

, metabolism

and

complex

behavior

al traits

, and may advance

discovery and development of molecular biomarkers for ASD.

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INTRODUCTION

Many

diseases

are associated with

informative

metabolic signatures

biomarkers

that

enable

diagnoses, predict disease course, and

guide

treatment

strategies

In contrast, a

utism spectrum

disorder (ASD) is diagnosed based on

observational

evaluation

behavioral symptoms,

including reduced social interaction and repetitive/stereotyped behaviors

(

)

The average age of

ASD

diagnos

is is

between

4 years

old

(

)

, at which time children can receive behavioral

therapy

the gold standard

treatment.

Because

earlier diagnosis

imp

roves

efficacy of behavioral

therapies

(

)

, molecular biomarkers represent an attractive approach

for

identif

ying

‘at

risk’

populations

and may

aid

deve

lopment of

personalized

therap

ies

This prospect

increasingly

important given the rising rate of

ASD

diagnoses

, which currently stands at

up to

1 in

children

in the United States

(

)

, with no FDA approved drugs for core

behavioral

symptoms

Metabolic

abnormalities have been

reported

in ASD

(

)

, though most have measured onl

small subset of metabolites and many outcomes have not reproduced between cohorts.

Mitochondrial d

isease, which heavily

influences systemic metabolism,

estimated to be higher

in ASD compared to controls (5% vs ~0.01%)

(

)

and

genes crucial for

mitochondrial

function

are risk factors for ASD in humans and

rodent models

(

)

The metabolic abnormalities

associated with mitochondrial dysfunction

in ASD

affect cellular energy, oxidative stress, and

neurotransmission in the gut and the brain

(

–

)

Othe

metaboli

profiles

ASD

implicate

romatic and phenolic metabolites

including

derivatives of

nicotinic, amino

acid, and hippurate

metabolism

(

–

)

Various amino acids are detected at differential levels across studies and

across sample types, but any consistent

patterns are difficult to

discern

(

–

)

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Some of the discrepancies between these studies are likely due to differences in

sample number

tissue analyzed

and methodology.

Other sources of variability include differences in

environmental

factors, such as

diet and gut

bacteria

which

differ

between ASD and TD

populations

(

–

)

. Diet is a major source of

circulating

metabolites

, impacting

the metabolome

directly or indirectly

through

chemical

transform

ation

by t

trillions of

gut

microbes

the

microbio

which

has been proposed

modulate complex behaviors in animal models and

humans

(

–

)

Such p

roposed environmental modulators of ASD

may integrate with genetic

risks to impact behavioral endpoints through the actions of small molecules produc

ed in

peripheral tissues outside the brain.

Herein

present

comprehensive

comparison of

an extensive

panel of

identified

metabolites

human

plasma an

feces

from a large cohort of

matched

ASD and

children

We identified

differential

levels

metabolites ranging from

hormone

, amino acid

, xenobiotic

, and l

ipid

many of which

correlate

with clinical

behavior

and GI

scores

To our knowledge, this is the first

study to concurrently evaluate paired intestinal and systemic metabolomes in a high

powered

analysis with a large number of identified metabolites, allowing direct associations between

metabolites previously highlighted

in ASD samples

and

discovering new metabolites of interest.

These findings

support the emerging concept of

evaluating non

behavioral features

in the

diagnosis of ASD

and its GI comorbidities

METHODS AND MATERIALS

Participants:

Samples for this study

, age

d 3

12 years old,

were collected through the UC Davis

MIND institute

(

)

. ASD diagnosis

was confirmed at the MIND Institute by trained staff

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using the Autism Diagnostic Observation Schedule (ADOS), and the Autism Diagnostic

Interview

Revised (ADI

R).

ubjects were diagnosed prior to 2013 based on DSM IV. Typically

developing (TD) participant

s were screened using the Social Communication Questionnaire

(SCQ). Participants in the TD group

score

within the typical range, i.e. below 15, on the SCQ

and above 70 on the

Mullen Scales of Early Learning (MSEL) and Vineland Adaptive Behavior

Score (VAB

S). Ninety

seven of the participants, who also provided the stool samples, completed

an additional evaluation to determine gastrointestinal (GI) symptoms. GI status was determined

using the GI symptom survey (GISS), based upon Rome III Diagnostic Questionn

aire for the

Pediatric Functional GI Disorders

(

)

and described in detail elsewhere

(

)

ee Supplemental materials

for additional methods

RESULTS

Plasma and Fecal Metabolomes Differ between ASD and

Plasma samples from 130 ASD and 101

children

were analyzed along with fecal samples

collected from a subset of these same ASD (n=57) and

individuals

(n=40) (Fig

ures

)

amples

and metrics of behavioral and GI scores

were

obtained from the

UC Davis MIND

institute

(

)

he ASD group

was stratified

into subsets of children with

GI symptoms

(ASD+GI) or without

GI symptoms

(ASD

GI)

explore

potential

effects

comorbid

intestinal

dysfunction in ASD

(

40 out of 130 ASD

samples were +GI)

. This stratification was based on

symptoms associated with ASD including diarrhea, constipation, and irritable bowel syndrome

like symptoms

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amples were analyzed by the globa

l metabolite panel (

plasma

and fecal samples) and the

complex lipid panel

(

plasma

samples

) by Metabolon

, Inc.

(Durham, NC)

which

identified

a total

panel of 1,611

plasma

and 814 fecal metabolites

(Table

Overall, we

discovered that

310

metabolites

were differentially abundant between the ASD and

groups

in plasma

and 112

metabolites

were

differentially abundant in feces

(Fig

ure

S1C, and

Tables

)

Using a quantitative assay for a targeted panel of metabolites

we observed a

high correlation

between relative abundance and

precise

concentration

(Fig

ure S

)

Overall, t

hese data

expand

on previous evidence

that the metabolomic profile

ASD

and

population

display

differences

not only in the gut compartment, but also in

circulation

, which

may

affect the levels

of metabolites

throughout the body,

cluding

the brain

(

)

appreciate the biological relevance of

differen

metabolomes

between ASD and

individual metabolite

were integrated

into

biochemical pathways

for

pathway enrichment

analysis

, revealing

the

degree of change

within each

Here, we identified large scale changes

mostly

lipid, xenobiotic

and nucleoti

de pathways

associated with

diverse

physiological

process

(Fig

ures

)

We then used

Random Forest

machine learning analysis

determine if

metabolite profiles can unbiasedly predict whether the sample came from an

ASD

and TD

donor

. Overall, the modest predictive accuracy of this machine learning approach was

68% for plasma and 67% for feces.

o test whether

focusing on

the

most

discriminating

metabolites

would

improve th

prediction

, we repeated the Random Forest analysis using the

top

30 metabolites

and found that the predictive accuracy

for plasma

improved slightly

to 69%

when

using all ASD samples

and

to 73%

when using only ASD

GI samples

For fecal samples, the

predictive accuracy improved

to 75% using all ASD and to 73%

using only ASD

GI.

The top 30

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metabolites

calculated b

y measuring the mean decrease in accuracy of

the

machine learning

algorithm

are useful to describe the

strongest

drivers of overall metabolic differences between

ASD and TD populations. These most dis

criminatory metabolites were

primarily

from the

lipid,

amino acid,

xenobiotic, and

cofacto

vitamin

super pathways

(Fig

ures

)

Several

of these

metabolites

have been previou

ly linked

to ASD

such as

steroids, bile acids,

acyl

carnitines

, and

nicotinamide

metabolites

(

–

)

Further, m

ultiple molecules known to be produced or

manipulated by the gut microbiota also feature

prominently, including 4

ethylphenyl sulfate

(4EPS

which is elevated in a

n ASD

mouse model,

and

indolelactate

, a

microbe

derived

trypt

ophan metabolite

(Fig

ure

)

(

)

The

two most discriminatory molecules in plasma

(Figures

)

and

feces

(Figures

) are

depicted

etabolites

correlating

the strongest

with these discriminatory metabolites are closely related on a structural and metabolic level

(Figure

1L).

Global

Metabolite Levels Correlate with

Clinical Behavioral Scores

Using

clinical

metadata

for

ASD

individual

, we

correlate

the levels of

individual

metabolite

to the verbal, social

and nonverbal scores of

standard diagnostic measures

the

Autism

Diagnostic Interview, Revised (

ADIR

)

a parent questionnaire,

and

the cumulative A

utism

iagnostic

bservation

chedule

severity score

(

ADOS

SS)

, conducted by trained health

professionals

(

)

(

Table

)

group

correlations between

the

behavior metrics and

entire

metabolite pathways

were calculated

(Figure

found that v

erbal and social scores

primarily correlate with lipid

metabolism

pathways

and

that

onverbal

scores have

the fewest

correlations

ADOS

SS correlated

with

diverse metabolite pathways, including

amino acid

and

food

plant component

pathways

, which may be

partly

due to

the

diverse array of symptoms

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integrated into

the ADOS

score

Additionally, when stratified by severity of behavior

score

we observe modest

clustering according to

plasma

metabolites correlated to the dis

order

between the

top and bottom quartiles of behavior severity groups for verbal, social, and ADOSS

SS metrics, but not for nonverbal

, and less so with fecal samples

(Figure

These

correlations

support

the involvement of lipid, amino acid, a

nd xenobiotic metabolism in the

etiology of ASD,

as previously described

(

)

, and

reveal new candidates for

ASD

biomarkers

that correlate with symptom severity

Transfer of ASD Fecal Microbiota into Mice is

Accompanied by Metabolic Signatures

Since microbial metabolites ranked highly in the

Random Forest

machine learning analysis

, w

tested if any of the observed metabolite differences in humans could be transferred to mice via

fecal microbial transplant. We

selected 4 male donor samples from

each of

the ASD and

group

and colonized

3 male

germ

free

mice per donor for three weeks

before collecting

plasma and fecal samples for metabolite profiling and bacterial DNA sequencing

(Figure S2C

S2D)

, respectively

Global metabolomic analysis revealed

that

olonized mice

modestly

cluster

by donor and

group

when statistically significant metabolites are considered in PCA analysis

(Figure

S2E

selected the human

donors based on 4EPS

levels

(Figure S2

)

due to

its

involvement in an ASD mouse model

(

)

and

dysregulation of similar phenolic compounds in

human ASD

(

)

4EPS is not produced by the host and is strictly a bacterial

metabolite

(

)

. Surprisingly, we observed

4EPS

levels

a bimodal distribution in mouse

samples (Figure

S2H

In spite of the surprising results with 4EPS,

many of t

he metabolites

with the highest fold change and lowest

value are

indeed

other

phenolic molecules such as

metabolites of hippurate, tyrosine, and diet

derived phenols

(Figure

Table S5).

hile

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preliminary,

these studies

reveal

microbiome

mediated

effects on xenobiotic pathways and

phenolic

metabolites

dysregulated in ASD.

Lipid and Xenobiotic Metabolites

Correlate

with GI Symptoms

Considering the prevalence of intestinal issues in ASD,

dependent

etabolite

analysis

between

individuals

may

provide

useful

insights

Accordingly,

we found a total of 87 and 2

metabolites in plasma and feces, respectively, that were differentially abundant within

the

ASD+GI compared to

ASD

individuals

(Figure

At the pathway level

, we

found

differences

free fatty acid

and xenobiotic metabolites

in the food component and plant

pathways

between ASD+GI vs ASD

plasma

(Figure

)

with

no broad

dependent

pathway alterations in fecal samples

(

Table S2).

In plasma,

free

fatty

acids

multiple chain

length

re lower in ASD+GI

compared to ASD

GI samples

, including monounsaturated,

saturated, and polyunsaturated fatty acids (PUFAs)

(Figure

PUFA

are anti

inflammatory

and lower levels of these fatty acids may contribute to

GI dysfunction directly

(

)

Several metabolites from the food component and plant pathway also

discriminate ASD+GI from

ASD

, such as

lower circulating

piperine metabolites

ASD+GI plasma samples

(

Figure

)

lower level of piperine

metabolites

was

also

associ

ated with

higher

ADOS

(Fig

ure

) and worse nonverbal scores

(

Table

Observed alterations to piperine metabolite levels

are

supported by the fact that

oral administration of

piperine has been

successfully

used as a

treatment in preclini

cal ASD

model

, presumably

due to

its antioxidant quality

(

)

While these

correlations are interesting,

the implications of

correlating an

altered metabolome and GI

symptoms in ASD remain to be determined.

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Steroid H

ormone Levels

are Elevated in ASD

Multiple

human

ASD

studies

have examined the levels of specific steroid metabolites within

androgenic, pregnenolone and progesterone metabolism, with some finding

aberrant

levels,

positive correlations with ASD

severity

, and behavioral improvement

following

treatment

lower levels

of certain

hormon

(

–

)

On the other hand, in a recent clinical t

rial

ASD

childre

given an

antioxidant treatment, levels of

pregnenolones and androgens increased and

correlated with improved behavior

(

)

In our dataset

, we found

robust

increase

most

every

detected

metabolite within the

pregnenolone,

androgen, progesterone

and corticosteroid

pathways

the

plasma of ASD children (Fig

ures

Table

Similarly,

some

androgenic

steroid pathway

metabolites

re elevated in ASD fecal samples (Fig

ures

Table S2

). This is

a strong indicator that the physiological pathways associated with

the downstream metabolism of

chole

terol

are significantly altered between ASD and

populations

(Figure

)

There does

not appear to be a global change in steroid meta

bolism, as most primary bile acid

and sterol

tabolites

re unaffected (

Table

e observe

some

elevat

ion of

these hormone

levels

independent of sex

, which is notable considering the male

bias in ASD

, reflected in our

primarily male sample

set

15% female)

(

)

(

Fig

ure S3

S3B

Because a cluster of

our

samples

are from older individuals

in the ASD group, and

to account for

age

dependent

increases

in androgen

we stratified by age

and

still

observed

heightened

androgenic and pregnenolone

metabolite

levels in ASD

sub

populations

(

Figure

Taken together, these data indicate that

steroidal hormone

metabolism

may be

altered in the ASD population relative to

samples and

that these differences are n

ot driven

sole

by sex or age differences in our cohort.

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Lipid Metabolite Levels

Differ

in ASD

Lipids are crucial for energy storage, cellular membrane integrity and cell signaling. They play a

variety of roles in the central nervous system, and their

dysregulation

has been linked to

ASD

(

)

hospholipids have been measured at lower levels, while long chain fatty acids

are

reportedly

elevated but PUFAs have been measured at both higher and lower levels

depending

on the cohort

(

)

Here,

we performed an

untargeted

, quantitative

metabolite

analysis on

complex lipid

he concentration

999 lipids were quantified

, and 13.7% of these were

significantly

different

in the ASD samples, with 4.6% increased and 9.1% decreased compared to

samples.

Many of these differentially abundant lipids included phospholipids, cholesterol

esters and glycerolipids.

In general, shorter (14

18 chain length) saturated fatty acids wer

less

abundant

in ASD throughout the lipid classes

(Figures

C, S

)

PUFA

lipid

levels were

also

different

, with elevations

in diacylglyce

rols and free fatty acids, and

an enrichment

of the

18:2

(linolenic)

chain length in most lipid classes

(Figure

ecal

samples generally

trended in the opposite direction from plasma

in PUFA lipids

(Figure S4

Table S2

Intriguingly, m

ultiple lipids with

linolenic

and

linoleic

(

18:3

)

chains we

re correla

ted

with social behavior (Tables

and

PUFA lipids containing linolenic and linoleic acids are

precursors to the important PUFAs (arachidonic 20:4 and docosahexaenoic 22:6 acids) for brain

development,

function,

and

structural i

tegrity

(

)

Future studies are needed to determine if

these specific changes in lipid

levels

contribute to ASD symptoms.

ASD Correlates with

Cellular Energy and Oxidative Stress Metabolite

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Many lipids are

markers of

mitochondrial and oxidative stress

offering a snapshot into cellular

metabolic state

. These markers include

acyl

carnitines

which

have been

highlighted

various

ASD studies

and are established indicators of

mitochondrial dysfunction

(

–

)

cyl

carnitines

are formed

to allow transport

of lipids

across the

mitochondrial membranes for beta

oxidation

, and abnormal levels of these conjugated lipids

accumulate to higher levels with a

decrease

in beta

oxidation

Interestingly, high levels of short

acyl

carnitines are found in

rodent

mode

where

ASD

ike

behavior

are

induced with the short

chain fatty acid

valproic and propionic acids

(

)

We fou

differential

lev

els of various acyl

carnitines

in ASD, creating a pattern of more

abundant short chain acyl

carnitines and less abundant long chain acyl

carnitines in

the ASD

samples

compared to

samples

(Fig

ure

)

Acyl

carnitines a

re positively

correlated with

more severe social

defects

effect

driven by

stru

tures

with

horter moieties (C2

C14) (Fig

ure

Table

)

. In fecal samples, a

cetylcarnitine (C2) and free car

nitine we

re elevated in ASD

(Figures

and

were highly

discriminatory

(Figure

Other mitochondrial markers

both plasma and feces

also

differentially abundant

ASD

and are summarized in

Figure

D along with

markers of

phospholipid

metabolism

, which occurs largely in the mitochondria

and

was

significant

ly altered

fecal

samples

(Fig

ures

Additionally

, the 5

plasma

metabolites most

positively correlated with ADOS

SS are all involved in

these

cellular energy

pathways

(Figure

S4D

Table

). These observed differences in energy markers and lipids could

have a

neurodevelopmental effect during

periods

when

the

high lipid

and energy requirement

the brain is crucial

(

–

)

, and alterations to levels of tricarboxylic acid (TCA) cycle

intermediates have been observed in human ASD prefrontal cortex samp

les

(

)

Such d

ects in

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cellular metabolism

pport the theory

that mitochondrial dysfunction

may not only be comorbid

with ASD

but also a potential

contributing factor, as suggested by numerous previous reports

(

)

mino acid

signatures

oxidative stress and mitochondria

l function

are

also

present

in our

dataset. Dysregulated amino acid degradation, homeostasis, and

import into the

brain ha

been

implicated as a cause of neuronal stress in ASD, and supporting metab

olomic data has shown

perturbations of various amino acid pathways

, such as glutamate, methionine, glutathione, and

gamma

glutamyl metabolites

(

)

, which exhibit

differences as

well (Figure 4E)

also demonstrate correlations between pathways

oxidative stress

(

cysteine, methionine, SAM and glutathione

pathways)

with

the ADOS

(Figure

ome of

these molecules we

re found in higher levels in the ASD feces and at lower levels in the ASD

plasm

, such as hypotaurine

(Figures

), which might indicate altered

fecal

production

excretion

or differential uptake into the plasma potentially through vari

intestinal permeability.

Similar to

hypotaurine,

levels of its precursor,

taurine

were significantly increased in ASD fecal

samples, although not altered in plasma (Figure S4E and S4F).

Taurine

plays

many roles

throughout the host, and

has previously been measured at altered levels in ASD, although with

little consensus

(

–

)

Hypota

urine and taurine

deficiency has

been

shown to lead to defects in

cell

differentiation

in the brain

(

)

and their dysregulation could

alter neuronal signali

(

)

xidative stress

glutathione pathway

precursors, gamma

glutamyl amino acids

are

relevant to

ASD

through their

influenc

e on

levels of neurotransmitters such as gamma

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aminobutyric acid (GABA)

(

)

, which is

widely thought to play a role in ASD

. In a recent

ASD study,

an experimental treatment that led to increased

gamma

glutamyl AAs and other

redox pathway metabolites correlated with improved behavior metrics

in children

(

)

lmost

every gamma

glutamyl amino acid is negatively correlated with ADOS

(Fig

ure

also

observe

perturbations

in the urea cycle, which process

the amino

group of amino acids for

excretion in urine

(Figure S4G

S4H

)

Abnormalities in this pathway can

be indicative of altered

amino acid degradation

observed in ASD

and can

lead to

neuro

tox

ic accumulation of nitrogen

containing compounds in the bloo

(

)

Together, these

results corroborate and extend a

growing body of research into altered mitochondrial metabolism and oxidative stress in ASD.

Differential Phenolic Xenobiotic Metabolite Levels

in ASD

henolic m

etabolites

, a diverse structural class

comprised of

thousand

s of molecules

containing

phenol

moiety

ome from dietary ingredients or from biotransformation of aromatic amino acids

by the gut microbiota. In their free phenolic forms they can be readily absorbed through

intestinal tissues; however, microbial modif

ication of these molecules can significant

alter

their

absorption, bioavailability, and bioactivity

(

)

, leading to various benefits or harm to the host

(

)

In fact, altered levels of phenolic molecules

have

been

hig

hlighted

many

ASD

metabolomic studie

(

–

)

However, a consensus of enrichment

or depletion o

f these metabolites across ASD vs

groups has yet to be reached, and most

studies

have

only

measured

small

subset of this structural class

of metabolites

observe

altered levels of

phenolic metabolite

belonging to several

interrelated pathways,

including tyrosine, benzoate, and food component and plant metabolites

(Fig

ure

ome

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molecules

such as

homovanillate and tyramine,

are involved in neurotransmitter metabolism

(

)

Oth

include

hippurate derivatives

which we

measured

lower

levels

in the ASD

plasma

samples

(Figure

Phytoestrogens such as daidzein, genistein, and equol derivatives

are elevated

in our ASD plasma samples

(Figure

These metabolites

have purported health

benefits but are also known disruptors of endocrine signaling

(

)

Additionally,

we detected

altered levels of

molecules

with

structural similarity to

para

cresol sulfate,

a toxic molecule

elevated in the urine of young ASD children

(

)

. These

include

, among others,

ethylphenyl sulfate

(

4EPS

)

, 2

ethylphenylsulfate,

cresol derivatives, 4

allylpheny

l sulfate,

and 4

methylbenze

sulfonate

, the latter of which was

elevated

a remarkable

fold

in a small subset

of ASD samples

(

Figure 5B

5D,

Table

Some

changes

in the levels of

these

phenolic

molecules

are

also

observed

in animal models of ASD

(

)

. P

reviously

4EPS was

observed

at a

fold

elevated level

in the maternal immune activation mouse model, and daily

administration of synthetic 4EPS to wild type mice was sufficient to induce an anxiety

phenotype

(

)

Here, i

n ASD plasma samples,

4EPS

levels

were

increased

6.9

fold

(

Figure

Other phenolic metabolite levels correlate with 4EPS in ASD samples, including

acetylpheny

sulfate,

derivative of 4EPS

, and others

(Fig

ure

, S5A

S5B

)

These observed

alterations in

phenolic molecules

, combined with

mounting evidence from

studies

suggest that

phenolic

structural metabolites

may

play a role in ASD

DISCUSSION

Changes in the metabolome have been linked to a number of

neurodevelopmental and

neurodegenerative disorders

(

)

. The

current

study

includes

comprehensive

profiling of

the

metabolites from

plasma and

97 matched

fecal samples from

ASD and

individuals

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who have been extensively behavior tested

We observed that

specific

individual

metabolites and

metabolic

pathways in

lipid, amino acid, and xenobiotic metabol

ism

were altered between ASD

and

groups

and that many of these same

molecules and

pathways correlated to severity of

ASD

behavioral

scores

The core findings are summarized in Figure 6.

Various

teroid hormone

metabolite levels

were elevated

in ASD sample

PUFA levels, and short chain acyl

carnitines

were generally elevated in ASD plasma, while saturated fatty acid levels and long

chain acyl

carnitines were decreased,

contributing to

general

picture of dysregulated cellular energy and

oxidative state

, with potential connections to

mitochondrial dysfunction in ASD.

Some of the

lipid and xenobiotic metabolites also showed interesting changes w

ithin the ASD group when

stratified by the presence of GI symptoms.

Finally, m

any phenolic metabolites, derived largely

from host and bacterial metabolism of amino acids, plant polyphenols, and other food

components were

detected

at differential levels in

plasma and feces

between the comparison

groups

ASD

diagnosed by behavioral tests,

with

extensive

heterogeneity of symptoms, severity, and

etiology

between individuals

and little consensus on molecular mechanisms

This enigmatic

spectrum

has a strong

but complex

genetic basis, with

hundreds

reported

risk genes

(

)

The

contributions of risk alleles to behavior is a vast and active area of research

. In addition to

gene

tics

, understanding

altered metabolite levels in blood, feces, and brain

of ASD

individuals

may prov

ide a glimpse into physiologic aspects of the disorder and hold the potential

to advance diagnosis and/or stratification of sub

populations of ASD.

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Interestingly,

ultiple mouse models support the notion that gut metabolites

are

associated with

brain development and

function

(

)

. Some metabolites are closely linked with

neurological disorders, either

to positive or negative outcomes

(

)

Several

exa

mples have

been

reported

of gut metabolites entering circulation and directly affecting the brain

as well as

cases where metabolites stimulate pathways in the gut, immune, or autonomic nervous system

and exert changes to

the

brain and

behavior

(

–

)

Comprehensive metabolic profiling in

human

and animal model

provide

insight into the molecular status of disease and how genetic

factors and environmental

risks

interact.

Deeper

analysis of

our

dataset

along with

additional

studies

with future

empirical studies to validate the relevance of

our

observations, could

illumin

ate

aspects of

ASD

pathophysiology

. The

intriguing

correlations between ASD behavior

altered levels of

fecal and plasma

metabolites,

and

GI symptoms contribute to the concept that

ASD

may be viewed as a

whole

body

condition, and argue for increased inves

tigation into

peripheral aspects of disease that may lead to advances in diagnosis and improved stratification

of ASD populations.

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ACKNOWLEDGMENTS

This work was supported by

grants from

Autism Speaks

(Grant #7567 to P.A., A.F. and S.K.M.)

;

the Johnson Foundation (to P.A

;

the Brain Foundation (to P.A

;

Genome, Environment,

Microbiome, and Metabolome in Autism (GEMMA)

825033 to AF

)

; Axial

Biotherapeutics

(to

S.K.M.)

; and

the National Institutes of Health (

HD090214 to P.A.

) and (

MH100556

to S.K.M.)

We would like to thank the MIND Institute study staff, and the dedication and commitment of

the families who took part in these studies is gratefully acknowledged.

Declaration of Interests

A.S.C., D.H.D

and S.K.M.

financial interest

in Axial Biotherapeutics.

A.F. has financial

interest in Alba Therapeutics.

G.M.P. and M.C.C. are employed by Axial Biotherapeutics.

B.D.N., M.D.A., G.S., D.R.R., and P.A. report no financial conflicts of interest.

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FIGURE TITLES AND LEGENDS

Figure 1. Plasma and Fecal Metabolomes Differ between ASD and TD and Global

Metabolite Levels Correlate with Clinical Behavioral Scores

B) The number of significantly elevated and decreased metabolites (pval<0.05) in ASD

samples compared to the TD contr

ol group by ANOVA contrasts in plasma and feces,

respectively. Samples are stratified by all samples or samples without or with GI symptoms (

GI,

+GI).