Derrick MacFabe M.D. 1 b Fermentation Products of the Gut Microbiome: Bench to Bedside Implications for Autism Derrick F. MacFabe M.D. (Assist. Professor and Director) The Kilee Patchell-Evans Autism Research Group Depts. of Psychology and Psychiatry (Div. Develop. Disabilities) Schulich School of Medicine and Dentistry, Lawson Research Institute, University of Western Ontario, London, Canada ([email protected]) b KPEARG Website: http://psychology.uwo.ca/autism.htm Publications/webinars Nature of Things Autism Enigma: http://www.cbc.ca/natureofthings/episode/autism- enigma.html DISCLAIMER Research depicts studies using rodents with EEG electrodes/drug ports to measure brain activity and behaviour, with studies based on extensive biochemical and tissue culture research Similar to human patients undergoing workup for surgical treatment of epilepsies Research ethics in strict accordance with Canadian Council on Animal Care and University of Western Ontario Animal Use Committee This basic science research in no way is intended to support any treatment claims by any groups not medically sanctioned by experienced physicians practicing evidenced based medicine in autism spectrum and related disorders. NO CONFLICTS DECLARED OVERVIEW Clinical Presentation of Autism Spectrum Disorders- a whole body disorder? A Family of Disorders- Multiple Causes- Final Common Pathway Genetics of Autism Environmental Risk factors- infection Neuropathology of Autism (altered brain development and neuroinflammation) Autism as a Metabolic Disorder-A Brain in an Energy Shortage The microbiome- the “Inner Rainforest” Antibiotics and the Western Diet- Clear Cutting the “Inner Rainforest” Can infectious agents control behaviour? Are the microbes in charge? Kilee Patchell-Evans Autism Research Group-multi-disciplinary- gut link to autism Using Animal Models to Study ASD’s -repetitive/ impaired social behaviour -brain electrical activity -neuropathology -immune/metabolic/gene activation Autism as the “Diabetes of gut fatty acid metabolism” Special populations- ASD with GI symptoms/Somali expatriates Future Directions in Autism Research and Treatment Autism – A Brain Disorder of Repetitive Movement, Restricted Interests, Sensory Sensitivity and Impaired Socialization Originally 1:10,000 (1950’s) Now 1 in 90 persons (males>females) Abnormal Social Interaction- object fixation Speech and Language Difficulties Repetitive Stereotyped Movements Self-Injurious Impulsive Behavior Sensitivity to Sensory Input Savant Syndrome (rare)/restricted interests Regression/ variable course in some patients “picky eating” carb craving Comorbidities: Seizure disorder Gastrointestinal dysfunction Immune/metabol. abnormalities CNS/GI* Mitochondrial disorder/dysfunction Genetic< 5% Genetic/environmental interactions? Enlarged Brain Size Increased Neuronal Density Altered Cell Migration Seizure Disorder White Matter Disorder Glial/microglial Changes Neuroinflammation (Impaired Neurodevelopment and Cortico-cortical processing) Systemic Changes Immune System Gastrointestinal System Metabolic Disorder Detoxification Systems (glutathione) Genetic Factors Neurotransmitter Growth Factors Cell-cell Interaction Sex Linked (Fragile X) Metabolism (carnitine synthesis) Environment Metals Hydrocarbons Infectious Drug (valproate) Diet- Wheat Casein Allergy Carbohydrate? Hormonal Sex Hormones Oxytocin Vasopressin AUTISM
Transcript
Derrick MacFabe M.D.
1
bFermentation Products of the Gut Microbiome:
Bench to Bedside Implications for Autism
Derrick F. MacFabe M.D. (Assist. Professor and Director)
The Kilee Patchell-Evans Autism Research Group
Depts. of Psychology and Psychiatry (Div. Develop. Disabilities)
Schulich School of Medicine and Dentistry,
Lawson Research Institute, University of Western Ontario, London, Canada ([email protected])
b
KPEARG Website:
http://psychology.uwo.ca/autism.htm
Publications/webinars
Nature of Things Autism Enigma:http://www.cbc.ca/natureofthings/episode/autism-
enigma.html
DISCLAIMER
Research depicts studies using rodents with EEG electrodes/drug ports to measure brain activity and behaviour, with studies based on extensive biochemical and tissue culture research
Similar to human patients undergoing workup for surgical treatment of epilepsies
Research ethics in strict accordance with Canadian Council on Animal Care and University of Western Ontario Animal Use Committee
This basic science research in no way is intended to support any treatment claims by any groups not medically sanctioned by experienced physicians practicing evidenced based medicine in autism spectrum and related disorders.
NO CONFLICTS DECLARED
OVERVIEW
Clinical Presentation of Autism Spectrum Disorders- a whole body disorder?
A Family of Disorders- Multiple Causes- Final Common PathwayGenetics of AutismEnvironmental Risk factors- infectionNeuropathology of Autism (altered brain development and neuroinflammation)Autism as a Metabolic Disorder-A Brain in an Energy ShortageThe microbiome- the “Inner Rainforest”Antibiotics and the Western Diet- Clear Cutting the “Inner Rainforest”Can infectious agents control behaviour? Are the microbes in charge?
Kilee Patchell-Evans Autism Research Group-multi-disciplinary- gut link to autismUsing Animal Models to Study ASD’s -repetitive/ impaired social behaviour
Autism as the “Diabetes of gut fatty acid metabolism”Special populations- ASD with GI symptoms/Somali expatriatesFuture Directions in Autism Research and Treatment
Autism – A Brain Disorder of Repetitive Movement, Restricted Interests, Sensory Sensitivity and Impaired Socialization
Originally 1:10,000 (1950’s)Now 1 in 90 persons (males>females)
Abnormal Social Interaction- object fixationSpeech and Language DifficultiesRepetitive Stereotyped MovementsSelf-Injurious Impulsive BehaviorSensitivity to Sensory InputSavant Syndrome (rare)/restricted interestsRegression/ variable course in some patients“picky eating” carb cravingComorbidities:Seizure disorder Gastrointestinal dysfunctionImmune/metabol. abnormalities CNS/GI*Mitochondrial disorder/dysfunctionGenetic< 5% Genetic/environmental interactions?
The Genetics of Autism- Identical twin studies- 50-80% concordance - genetics and environment- Multiple Chromosomes- 2,3,7, 15, 16, 17 X- chromosome mapping-Multiple genes- brain development, neurotransmitters, language centres-Intercellular connections- Neurexins-Disorder of Gene expression (methylation, acetylation of histones)-Met Receptor Tyrosine Kinase &-Protein Kinase C beta 1 (brain, gut, immune)- at present 85-97% NO DEFINED GENETIC CAUSE-Oversimplistic to say one specific genetic cause-Epigenetics- interaction with genes/environment-“Spontaneous (?)” genetic mutation- Other genetic disorders where autism is associated
Fragile X**Angelman/Prader Willi Syndrome**Epilepsy- “tuberous sclerosis”* Rett Syndrome
Many environmental/infectious factors mimicGenetic transmissibility
(i.e. Tuberculosis, twins with same/different placenta)Genetic sensitivity to infection (similar pattern in ASDs!)
Environmental agents as gene “switches”
Neurodevelopment- “Lets Build a Brain”- Complex development
timing important- Many neurons die
Genetic (instruction)-EnvironmentInsults:Infection (virus)/inflammatory (IL-6)toxins (alcohol)/metals/drugs(valproate)Oxidative stress-Redox change- cell fate(germ cell-fetus-neonate)Cell to Cell Communication is Important in the organization of thedeveloping nervous system (programmed cell death and ordered cell migration)Reelin, neurexins , Gap junctions, see later……..environmental factors may alter neurodevelopment
Neuropathology of Human Autism– Neuroinflammation/mild cell lossOngoing chronic process throughout life of Patient (Pardo)
MG
MG RG
RG
NRL AUT
RG MG
Autism- A Disorder of Energy Utilization and Toxin Elimination
Oxidative Stress (Chauhan, James):Inflammation, impaired metabolismProcess similar to memory!!!Antioxidants- glutathioneFacilitators of mitochondrial function-carnitine, methylation-Methyl B12 (accessibility to CNS?)A mitochondrial disorder?(Mitochondrial DNA mutations- risk) Rossignol and Frye 2011
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Mitochondrial disease /dysfunction (Frye) energy dysfunction?Heterogeneous tissues affected/families/ complex inheritance
Cell wall- LPS, beta glucan- innate immunity“priming immune system & fat metabolismFermentation products of dietary carbohydrate- Short chain fatty acids*
Barriers, variable metabolism
Acquired/genetic (met receptor tyrosine kinase)
Timing of exposure
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The Human Microbiome Gut Microbiome- Complex Ecosystem- Alteration with Antibiotics
Small bowel microbial overgrowth- bugs in the wrong place
Fluffy little bunnies are cute,but not in Australia!
Consequences of Overfeeding
Yangtze River Delta Pollutionone third of population China
++ fertilizer“overfeeding the ocean”
Ecosystem crashJelly fish bloom
“the clostridia of the ocean?”
Colonization of Newborn with Hospital Acquired Antibiotic resistant florae, feeding or long term antibiotic treatment- Altered Microbiome Development of Agriculture and Animal Husbandry
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Development of Cereal Diet, Animal Domestication & Urban CultureCo-Evolution of Endogenous Florae? (J. Diamond/M. Pollan)Cultural Taboos with cattle/dairy
“Urbanization of Cattle”- Crowding, Corn and Chronic Antibiotics(clostridia, propionibacteria)
Obese vs. lean rats- switch gut flora at birth- switch body type!Long term antibiotics in rats change gut bacteria/fat metabolism
Intestinal pathology on a subset of autistic patientsAssociated with regressive onset and GI symptomsModerate inflammatory process (nonspecific?)NOT CAUSED BY VACCINES!!!! Cause????
Psssst…Dr. MacFabeFeed Us and Spread Us Around!!
Can Enteric Bacteria Affect Brain Development/Behaviour?
Clinical- Food Craving/Symptom Worsening/ GI symptomsGut changes (gluten/casein) poorly studied (antigenic mimicry)Early gut colonizers- alteration with antibiotics (increased incidence)Unique bacterial species (clostridials, desulfovibrio, bacteriodetes)“Leaky” or malabsorbtive digestive tract (impairment of barriers)Production of bacterial metabolites (fuel for brain)Effect on Brain development, physiology, behaviour, immune function
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Pathogen “Control” of Host Nervous System for Propagation
Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcal Infection (PANDAS)- (Swedo)
Associated with Group A Beta Strep infections”Clingy- OCD/Tick symptoms, relapsing remitting
Autoantibody to basal gangliaSimilar behaviour in family (sensitive population)
GM+, spore,Toxin A (enterotoxin),B (cytotoxin) binary?,biofilm (“hiding”)Severe- pseudomembranous
Colitis? Mild infections/carrier state?age of infectionFinegold- regressive ASDUnique bacterial populations
C. difficile
Carbohydrate Craving, Unique
OpportunisticBacteria
Diarrhea and Fecal Smearing in
AutismBehaviour facilitates growth and spread of autism implicated gut
pathogens (clostridials)?
Pathogen affecting host behaviour
Photo- M. Herbert
Clostridium Difficile – Epidemic
Genetics ARE important- Genetic mutations of infectious processes too!
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Autism in Somali Diaspora in North America
3% of general population, 35% of autism in some regionsAll conceived in Receiving Country- NOT Somalia
Large exposure to antibiotic/++ gastrointestinal infections
Propionic Acid- Neuroactive properties
Weak organic acid: lipid/water soluableUptake passive active (monocarboxylate receptors)G protein coupled receptorsIntracellular concentration (intracellular acidification)Unique CNS/GI immunological properties
Short Chain Fatty Acids – Propionic Acid (PPA)
Propionate:
Byproduct of bacterial metabolismClostridium, Propionibacteria (gut/acne)Desulfovibrio (Finegold)(butyrate, acetate)- short chain fatty acids
Common preservative of wheat and dairy products ( alone or Nabitor)Increased by ethanol, B12/biotin deficiency
Variable metabolism of propionate in population – Multiple mechanisms and multiple clinical presentationshares similarities with autism- underreported???
Role of diet, gut bacteria/barriers and “sickness” in propionate levels(other short chain fatty acids and metabolites)
•A Review of Propionic Acidemia:
• Part of a family of metabolic disorders (methylmalonic acidemia• propionyl CoA carboxylase, multiple carboxylase, biotinidase deficiency• Considerable polymorphisms (chromosome 3 and 13) – underreported• May be 10x more common (Asian, Mediterranean)• Elevated in other organic acidemia, biotin/B12 deficiency, alcohol• Developmental delay, seizure, movement disorder, GI disturbances.•Autism like behaviour• Acidosis/ propionate excretion may or may not be present•NB- difficult to measure PPA and metabolites
•A Review of Propionic Acidemia (cont):
•Mechanisms:
• Mitochondrial disorder leads to increased propionate/• propionyl CoA – mitochondrial toxin• Intracellular accumulation of short chain fatty acids leading to acidosis. • Increased nitric oxide, peroxide, impaired –SH, • NB-Carnitine depletion – mitochondrial uncoupling• Glutamate/dopamine 5HT release •lipoperoxidation (membrane damage)• Gene expression (Tyrosine OHase, enkephalins)• histone deacetylase inhibitor (gene expression)• “Sensitivity to metabolic stress”
Propionate leads to reduced antioxidant defences/PropCoA toxicityBiotin deficiency common in pregnancy (gut bacterial source)
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Rodent Model of Autism- Effects of Propionic Acid Administration(MacFabe et al, Behavioural Brain Research 2007),MacFabe, Microbial Ecology 2012 for Reviews
PPA
Autism Model –Propionic Acid (PPA)- behaviour/EEG
Effect immediate, transient (45min) but some permanent
PBS
Propionate Autism Model
-Injected into cerebral ventricles-NB buffered to pH 7.5- Reversible repetitive behaviour-Fixation on objects-Seizure +/behaviour cortex-Subcortical spiking
Automated Behavioural Monitoring (Ethovision):- Computerized long term quantification of movement- Combined with drug administration, brain electrical activity- Repetitive behaviour
Vehicle
Propionic Acid Autism Model
Total Distance Infusion Day 1 Low Dose
Time (min)
0 10 20 30 40 50 60 70 80 90 100 110 120
Dis
tanc
e To
tal (
cm)
0
200
400
600
800
1000
1200
1400
1600
PBS PPA
Intraventricular PPA- “ritual”
Hippocampal EEG- Repetitive motor loopNormal EEG
PPA induced kindling in Cortex,Hippocampus, CaudateNo effects seen with control treatments (PBS, propanol)Brain “remembers” exposure
Propionic Acid induces long convulsion- Acetate short (metabolized)
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Subcortical Spiking with PPA
Caudate spikes lead to limb dystoniaNormal Hipp, frontal spikingCaudate spike 500ms preceed CTXOnly PPA , no other groupsProduce Caudate eventNo kindling, immediateRole in movement disorder
encephalopathyautism??
Would be missed by traditionalSurface EEG leads in pts
0
2
4
6
8
10
Mea
n R
etro
puls
ion
Bou
ts Baseline Treatment
Retropulsion
Low Propionate
High Propionate
Propanol Sodium Acetate
PBS
*
0
3
6
9
12
15
Mea
n N
umbe
r of S
nake
Pos
ture
s
Snake Posture
Low Propionate
High Propionate
Propanol Sodium Acetate
PBS
*+
0
10
20
30
40
50
Mea
n Tu
rnin
g
Turning
Low Propionate
High Propionate
Propanol Sodium Acetate
PBS
##
Legend* = Significantly different from all control groups.# = Significantly different from low PA, propanol, PBS.+ = Significantly different from propanol and PBS
Propionic acid causes movementdisorder with caudate spiking
Caudate Spiking and Limb Dystonia Only Caused by PPA
Movement disorder effects of PPAMost sensitive
Social Behaviour (Ignoring/Mean Distance Apart)(Shultz et al. Neuropharmacology, 2008)
vehicle PPA
Effect apparent after one dose, reversible post metabolismReduced play behaviour
Social “Ignoring” of Normal Rat
PBS PPA
MacFabe et al; Behavioural Brain Research (2010)
PPA Rats Prefer “Favourite Objects” to other Rodents (MacFabe et al, 2010 BBR)
1 2 3 1 2 3Object:
40
30
20
10
0
Entri
es in
to z
one
Object Zone EntriesPPA PBS
1 2 3 1 2 3Object:
100
80
60
40
20
0
Dur
atio
n in
zon
e (s
ec)
Duration in Object Zone
1 2 3 1 2 3Object:
30
25
20
15
10
5
0
Sniff
Bou
ts
Object Sniff Bouts
A
B
C
B
C
PPAPBS
250
200
0
Dur
atio
n (s
ec)
Duration in Close Proximity
150
100
50
Proximity to rat Proximity to object
0
Perc
ent t
ime
Time Approaching Rat or Object40
30
20
10
Approaching rat Approaching object
A PPA PBS
NR NR
NO NO
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Long term effects- Ethovision- stereotypies/ object fixation
PBS-first trial PPA-first trial-nrl acquisition
PBS- reversal PPA-reversal- perseveration
Morris Water Maze- perseveration- Shultz et al, 2009
microinjections of propionate - very brief exposureAstrogliosis - prominent, hippocampus, cingulum, white matter Neuroinflammation (TNF alpha)Toxic or compensatory (neuroplastic response)
PPA can induce multiple genes implicated in learning, memoryaddiction, neurodevelopmentEnvironment influencing genetic expression!(overexpression- Anxiety, obsession?
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Control (PBS) PPA
Monocarboxylate I Transporter (fatty acid uptake)
Transporter in brain/ gut endothelium- developmentImportant in weak acid transport (SCFAs, lactate) and ketonesAlterations in fatty acid transport?(critical in neurodevelopment and cell signaling)
Is Propionate Cytotoxic? - Activated Caspase 3’(apoptosis)
cont High PA
PPA is not grossly neurotoxic in hippocampusmay be a neuroprotectant!- histone deacetylase inhibitionNeuroinflammation with little neurotoxicity
PPA Digital analysis
Interleukin 6PBS PPA
WM
Hipp
PBS Vehicle High Dose PPA
PPA causes increase anti Nitro-tyrosine immunoreactivity in hippocampal formation increases “oxidative stress”
Anti Nitrotyrosine Immunoreactivity- oxidative stress
Oxidative Stress Markers
μM
of M
DA&
HN
E/m
g pr
otei
n
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
2.5
PBSPPAHigh
nM
car
bony
l der
ivat
ives
/mg
prot
ein
*
**
lipid peroxidation marker protein oxidation marker
Glutathione System
Spec
ific
Act
ivity
nmol
/min
/mg
of p
rote
in
0
20
40
80
100
μM o
f tot
al G
SH
/mg
of p
rote
in
2
4
6
8
10
12
PBSPPAHigh
**
GPx GST GR GSH
***
Increased Oxidative Stress in PPA Autism Model(MacFabe et al Am.J. Biochem.Biotech.2008)
PPA increases oxidative stress markers and impairs Glutathione metabolism (sequestration?)-brain “sensitive” to broad spectrum of environmental agents(ie metals, xenobiotics)-similarity to evidence of metabolic dysfunction in ASD patients-broad effects- metabolic encephalopathy
O
OC
HO
CH3
HO
OH
OH
O
OC
OCO
H3C
CH3
O
O CH3
OCHO
O CH3
CO OH
OHOH3C
Cl OHH3C
OC OH
O
OH
OH
OC
OH3C
CHO
O
O CH3
Fatty acids (FA) in Autism
• Building block of lipids (low FFA)• Characteristic COOH functional group• Saturated (animals) and unsaturated ( plants)
(mono vs poly), (trans vs cis)• Odd or even numbered (C2 - C26)• Modified (hydroxylated, methylated,
dicarboxylated, etc)
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Where do fatty acids (lipids) come from?
• Body synthesis
• Diet:– Essential and non essential fatty
acids– Products of enteric bacteria
• Essential fatty acids– Body cannot make– omega 3’ and omega 6– Balance between ω3 and ω6
affect functions
Omega 3 (ω3) FA
Omega 6 (ω6) FA
Functions of Fatty Acids• Main functions:
– Energy storage– Structural components of cell
membranes– Act as signal molecules in many
metabolic processes– Abnormal fatty acid composition in
eradication/probiotics/MB12? Multi- system approach (metabolite measurement diagnostics)
Baseline T1 T2 T3 T4 T5 T6 T7 T8 --0
500
1000
1500
2000
2500
3000
3500
4000
4500
Mea
n To
tal D
ista
nce
Mov
ed (c
m)
Treatment Days
PBS PPA Buturate
TOTAL DISTANCE
* *
^
+
**
***
^^
^^
^^
*
Intraventricular infusion PPA and Butyric Acid induce increased locomotor activity in rats
(gut SCFA metabolites can alter behavior)
Butyric AcidPPA
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Average Startle
Trial type115 dB 73 dB 76 dB 82 dB no pulse
Ave
rage
sta
rtle
(mV
)
0
20
40
60
80
100PBS n = 30PPA n = 31
* **
Adolescent Behavioural Changes in Response to Early Exposure to PPA:Acoustic Startle
During first few days of life, rat pups are injected with sub cut PPA or PBS andBehaviourally tested as adolescents.
The amount that an animal is startled (“jumps”) in response to an acoustic stimulus is measured. PPA animals are more sensitive to stimuli – jump more – than PBS animals.- Reduced inhibition (i.e GABAergic dysfunction).
SCFA activate the transcription of TH gene
PPA and valproate induce tyrosine hydroxylase expression in PC12 cells(valproate modified SCFA- autism risk factor)
Via a CREB dependant mechanism (same in brain homogenate)Epigenetic control of catecholamine synthesis(Dopamine- movement, aggression, addiction)
Time-of-flight secondary ion mass spectrometry (ToF-SIMS)
Multiple analysis of metabolic intermediates/metals in CNSOxidative stress- nitrosylation,
altered lipid profiles (decreased cholesterol) and edema (Na and Cl) in White Matter- consistent with ASD
Compare with other Somali populations (US, Sweden), Saudi (urban/rural) and general population
Assistance ASDCARC, U. of Guelph, King Saud and Karolinska
Bacterial Ecology of Regressive Autism/ “Repoopulate”
Microbial ecology ofregressive autism
Reconstitution of “scaffolding”of key gut
microbial communitiesC. diff/ulcerative colitis
Tore Midtvedt- Karolinska- Grandfather of the Microbiomefollowing babies for health or disease- germ free animals
Nobel Symposium-Autism and Gut-Karolinska(Stockholm)-Midvedt,Nicholson, Forssberg, Reichelt, Bolte, Bienenstock, Falk-Ytter
Summary
Autism is a complex problem needing a multi-disciplinary approach
with modern brain research and microbiology techniques, much is available to rationally examine autism as a defined brain disorder
detailed study of behaviour in animal models necessary to determine cause and develop rational clinical studies and treatment
Factors in brain development- neural migration, embryonic cell deathtoxic environmental compounds (dietary and enteric fatty acids)role of diet, metabolism and gut microbiome (antibiotic exposure)
Gut metabolites can alter brain electrical activity, behaviour, Pathology, gene induction and cellular metabolism (mitochondria)Bacteria modulating host behavior! Western environment changing!!Variable exposure/breakdown in humans/microflorae
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Acknowledgements- Kilee Patchell- Evans Autism Research Group
Klaus-Peter Ossenkopp, Ph.D., Donald Peter Cain, Ph.D., Martin Kavaliers, Ph.D., (Department of Psychology (Neuroscience)- University of Western Ontario)
Fred Possmayer PhD. (Biochemistry, Obstetrics/Gynecology)Lisa Tichenoff, Kelly Foley MSc., Roy Taylor, Francis Boon, Soelaha Shams ,Melissa
Meeking, Andrew Franklin MSc., Jennifer Hoffman MSc., Jennifer Mepham , Raymond Thomas, PhD., Karina Rodriguez-Capote, PhD, Yalda Mohammad-Asef,
Short Chain Fatty Acid BacterialFermentation Products
Gap Junction Closure
Short-chain fatty acid fermentationproducts of the gut microbiome:implications in autism spectrumdisorders
Derrick F. MacFabe, MD*
Director: The Kilee Patchell-Evans Autism Research Group, Departments of Psychology (Neuroscience) andPsychiatry, Division of Developmental Disabilities, Lawson Research Institute, University of Western Ontario,London, ON, Canada, N6A 5C2
Recent evidence suggests potential, but unproven, links between dietary, metabolic, infective, and gastro-
intestinal factors and the behavioral exacerbations and remissions of autism spectrum disorders (ASDs).
Propionic acid (PPA) and its related short-chain fatty acids (SCFAs) are fermentation products of ASD-
associated bacteria (Clostridia, Bacteriodetes, Desulfovibrio). SCFAs represent a group of compounds derived
from the host microbiome that are plausibly linked to ASDs and can induce widespread effects on gut, brain,
and behavior. Intraventricular administration of PPA and SCFAs in rats induces abnormal motor movements,
repetitive interests, electrographic changes, cognitive deficits, perseveration, and impaired social interactions.
The brain tissue of PPA-treated rats shows a number of ASD-linked neurochemical changes, including innate
neuroinflammation, increased oxidative stress, glutathione depletion, and altered phospholipid/acylcarnitine
profiles. These directly or indirectly contribute to acquired mitochondrial dysfunction via impairment in
carnitine-dependent pathways, consistent with findings in patients with ASDs. Of note, common antibiotics
may impair carnitine-dependent processes by altering gut flora favoring PPA-producing bacteria and by
directly inhibiting carnitine transport across the gut. Human populations that are partial metabolizers of PPA
are more common than previously thought. PPA has further bioactive effects on neurotransmitter systems,
or a combination of these factors. There is a growing
interest in examining ASDs as a disorder of glial cell
function. Glial cells are of great importance in both the
developing and mature nervous system, particularly with
respect to cell�cell interactions during neural migration,
(page number not for citation purpose)
�THEMATIC CLUSTER: FOCUS ON AUTISM SPECTRUM DISORDERS
Microbial Ecology in Health & Disease 2012. # 2012 Derrick F. MacFabe. This is an Open Access article distributed under the terms of the Creative Commons Attribution-
Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
1
Citation: Microbial Ecology in Health & Disease 2012, 23: 19260 - http://dx.doi.org/10.3402/mehd.v23i0.19260
pesticides (43�45), and finally enteric metabolic products
of ASD-associated intestinal bacteria (46�53).
The ‘inner’ outer environment, the entericmicrobiome as a source of environmentaltriggers of ASDsThe human digestive tract is host to a complex array of
intestinal bacterial florae, coined the microbiome, which
outnumber host cells at least 10 to 1. The microbiome
produces an array of bioactive metabolic products
capable of entering systemic circulation. It is important
to note that the enteric microbiome and its metabolic
products are not static and can be altered throughout the
life cycle of the individual, particularly throughout the
first 18 months of life (54). The metabolic products from
the gut microbiome can have profound and dynamic
effects on host metabolism, immune function, and gene
expression in many organ systems, including the CNS
(46, 55�60). In addition, it is also important to consider
the recent impact of the alteration of the human
microbiome and its metabolites through the introduction
of the high calorie Western diet, coupled with the high
exposure of antibiotics and disinfectants to human
beings, animals, and plants, as a possible source of
environmental triggers of many diseases of increasing
Derrick F. MacFabe
2(page number not for citation purpose)
Citation: Microbial Ecology in Health & Disease 2012, 23: 19260 - http://dx.doi.org/10.3402/mehd.v23i0.19260
How are dietary and gastrointestinal factorsrelated to autism? � microbiome production ofenteric fatty acids as environmental triggersThere is a growing interest suggesting a link between
dietary and/or gastrointestinal factors and the worsening
and, in some cases, improvement of ASD symptoms, but
the mechanisms responsible for this remain elusive. As
indicated, SCFAs, such as PPA, are produced by many
gut bacteria by the breakdown of dietary carbohydrates
and amino acids, particularly from wheat products (97).
Of particular relevance are the Clostridia and Desul-
fovibrio, which have been proposed as infectious causes
of ASDs (64, 66). Clostridial species, a family of hetero-
PPA-induced intracellular acidification,increased oxidative stress, impaired antioxidantcapacity, and gap junction closureThe findings that PPA and its related fatty acids often
induce similar behavioral effects, whereas isomolar infu-
sions of propanol do not (46, 49, 50), make it tempting to
speculate that the carboxylate functional group and/or
acidic properties of PPA may be involved in its effects
on behavior. Once low-molecular-weight organic acids
are protonated in acidic conditions, they become more
lipid soluble and, thus, gain access to the CNS, either by
passive diffusion or active transport (77, 78, 80). Once
they enter the CNS, they may concentrate within cells
and induce intracellular acidification. Previous studies
have found that PPA- and acetate-related acidosis in rats
can alter social behavior (139). Furthermore, clinical
metabolic acidosis from varied etiologies in humans often
involves bouts of confusion and movement disorder
similar in some respects to those found in ASDs (140).
The effects of varying intracellular pH on cellular
physiology are broad. Notably, PPA-induced intracellular
acidification can inhibit mitochondrial function, which is
vital for normal metabolism of other fatty acids (141).
Intracellular accumulation of organic acids creates a
buildup of acyl-coenzyme, which interrupts metabolism
(141). PPA sequesters carnitine function and inhibits
CoA function and, thus, impairs mitochondrial metabo-
lism and energy production, leading to further decrease in
cytoplasmic pH via the accumulation of other organic
acids (131, 141). This is consistent with the impaired
mitochondrial metabolism in ASDs, including carnitine
deficiency, mitochondrial dysfunction, and systemic ele-
vations of nitric oxide metabolites (20, 46, 142). In
addition, valproate, an antiepileptic drug, structurally
related to PPA, and a known prenatal risk factor for
were observed. Specifically, when these acylcarnitines
were grouped according to chain length, acetyl carni-
tine (C2) was the major contributor to the increases ob-
served in short-chain acylcarnitines, while C16:0, C18:0,
C18:1, C22:0, and C22:1 were the major contributors
to the increases observed in long-chain acylcarnitines
(51, 53).
These unique acylcarnitine profiles are similar to those
obtained from blood samples of patients with ASDs (182,
183) which observed elevations in the levels of some long-
chain acylcarnitine species (14:1 and 14:2). Elevations in
14:1 and other long-chain acylcarnitines (16:0, 18:0, 18:1,
22:0, and 22:1) consistent with findings in our model.
PPA is thought to affect mitochondrial fatty acid
metabolism by binding to propionyl CoA and by
sequestering carnitine (86, 186, 187). Elevation in the
levels of these acylcarnitines indicates a potential meta-
bolic disturbance by PPA infusions to affect mitochon-
drial metabolism in manners consistent with a unique
acylcarnitine profile and a proposed environmental
induction of mitochondrial dysfunction and increased
oxidative stress in ASDs (51, 188).
Of particular interest, PPA is a known inhibitor of
mitochondrial function, through sequestration of carni-
tine and the production of propionyl CoA, a potential
cytotoxin (189, 190). PPA also inhibits the incorporation
of acetate during lipid synthesis, an effect which is more
pronounced in males (191, 192). This is interesting in
light of low cholesterol levels being central to many forms
of ASDs (178). Since PPA is metabolized through the
mitochondrial metabolic pathways and affects mitochon-
drial cardiolipin levels and membrane stability (51, 53),
we propose that excess exogenous PPA or its related
SCFAs overwhelm mitochondrial metabolism, thereby
causing mitochondrial dysfunction. Such mitochondrial
dysfunction is consistent with an increase in oxidative
stress. Evidence for oxidative stress has been demon-
strated in the rodent PPA model of ASDs by the increase
in nitric oxide-/hydrogen peroxide-producing activated
microglia, coupled with the elevation of oxidized proteins
and lipids, reduction of glutathione, and alteration of
phospholipid/acylcarnitine profiles in brain homoge-
nates, particularly in brain regions affected in ASDs
(46, 51, 53). Such findings are all consistent with those
obtained from patients with ASDs (110, 151, 152,
193, 194).
Impairment of carnitine metabolism from avariety of causes may be central to ASDpathogenesis and regressionAlthough the basis of the reduced blood carnitine levels
in ASDs remains unclear, we have made the observation
that there are diverse clinical conditions circumstantially
linked to ASDs and gastrointestinal dysfunction that
show disruptions in carnitine metabolism as a common
observation. There has been a growing interest in the role
of carnitine in brain physiology and disease (181),
particularly in brain GABAergic and astrocyte metabo-
lism (195). As carnitine is endogenously produced from
lysine and methionine, persons with defects in methyla-
tion pathways, common in individuals with ASDs (196),
would have impairments in endogenous carnitine synth-
esis. A particularly vulnerable subgroup would be the
recently discovered X-linked defect in the TMLHE
enzyme, responsible for the first step in carnitine bio-
synthesis, which is a risk factor for non-dysmorphic
autism in males (25). Collectively, these persons would
rely more on dietary sources of carnitine, critical during
periods of rapid development, and thus may be more
sensitive to factors that impair carnitine uptake from
the gut. It is known that carnitine is transported across
the gut�blood and blood�brain barriers via the Na�-
dependent organic cation/carnitine transporter 2
(OCNT2) (197). Carnitine transport deficits have been
implicated in colitis (198) and also may impair blood�brain barrier integrity, allowing non-neurotropic influ-
enza A virus to enter the CNS and inducing a neonatal
215. Sokol MS. Infection-triggered anorexia nervosa in children:
clinical description of four cases. J Child Adolesc Psychophar-
macol 2000; 10: 133�45.
216. Kerbeshian J, Burd L. Is anorexia nervosa a neuropsychiatric
developmental disorder? An illustrative case report. World J
Biol Psychiatry 2009; 10: 648�57.
217. Diamond J. Guns, germs, and steel. New York: W.W. Norton &
Company; 1997.
218. Foley KA, Kavaliers M, Ossenkopp K-P, MacFabe DF.
Prenatal exposure to propionic acid and lipopolysaccharides
produces developmental delay, anxiety-like behaviour, and
hyper-sensitivity to accoustic startle in adolescent rats. abstr.
International Meeting for Autism Research, May17�19, 2012,
Toronto.
*Derrick F. MacFabeDirector: The Kilee Patchell-Evans Autism Research GroupDepartments of Psychology (Neuroscience) and PsychiatryDivision of Developmental DisabilitiesLawson Research InstituteRoom 9244, Social Science CentreUniversity of Western OntarioLondon, Ontario, Canada, N6A 5C2Tel: 519 661 2111 ext 84703Email: [email protected]
Derrick F. MacFabe
24(page number not for citation purpose)
Citation: Microbial Ecology in Health & Disease 2012, 23: 19260 - http://dx.doi.org/10.3402/mehd.v23i0.19260
Unique acyl-carnitine profiles are potential biomarkersfor acquired mitochondrial disease in autism spectrumdisorder
RE Frye1, S Melnyk1 and DF MacFabe2
Autism spectrum disorder (ASD) has been associated with mitochondrial disease (MD). Interestingly, most individuals with ASDand MD do not have a specific genetic mutation to explain the MD, raising the possibility of that MD may be acquired, at least in asubgroup of children with ASD. Acquired MD has been demonstrated in a rodent ASD model in which propionic acid (PPA), anenteric bacterial fermentation product of ASD-associated gut bacteria, is infused intracerebroventricularly. This animal modelshows validity as it demonstrates many behavioral, metabolic, neuropathologic and neurophysiologic abnormalities associatedwith ASD. This animal model also demonstrates a unique pattern of elevations in short-chain and long-chain acyl-carnitinessuggesting abnormalities in fatty-acid metabolism. To determine if the same pattern of biomarkers of abnormal fatty-acidmetabolism are present in children with ASD, the laboratory results from a large cohort of children with ASD (n¼ 213) whounderwent screening for metabolic disorders, including mitochondrial and fatty-acid oxidation disorders, in a medically basedautism clinic were reviewed. Acyl-carnitine panels were determined to be abnormal if three or more individual acyl-carnitinespecies were abnormal in the panel and these abnormalities were verified by repeated testing. Overall, 17% of individuals withASD demonstrated consistently abnormal acyl-carnitine panels. Next, it was determined if specific acyl-carnitine species wereconsistently elevated across the individuals with consistently abnormal acyl-carnitine panels. Significant elevations in short-chainand long-chain, but not medium-chain, acyl-carnitines were found in the ASD individuals with consistently abnormal acyl-carnitinepanels—a pattern consistent with the PPA rodent ASD model. Examination of electron transport chain function in muscle andfibroblast culture, histological and electron microscopy examination of muscle and other biomarkers of mitochondrial metabolismrevealed a pattern consistent with the notion that PPA could be interfering with mitochondrial metabolism at the level of thetricarboxylic-acid cycle (TCAC). The function of the fatty-acid oxidation pathway in fibroblast cultures and biomarkers forabnormalities in non-mitochondrial fatty-acid metabolism were not consistently abnormal across the subgroup of ASD children,consistent with the notion that the abnormalities in fatty-acid metabolism found in this subgroup of children with ASD weresecondary to TCAC abnormalities. Glutathione metabolism was abnormal in the subset of ASD individuals with consistent acyl-carnitine panel abnormalities in a pattern similar to glutathione abnormalities found in the PPA rodent model of ASD. These datasuggest that there are similar pathological processes between a subset of ASD children and an animal model of ASD with acquiredmitochondrial dysfunction. Future studies need to identify additional parallels between the PPA rodent model of ASD and thissubset of ASD individuals with this unique pattern of acyl-carnitine abnormalities. A better understanding of this animal model andsubset of children with ASD should lead to better insight in mechanisms behind environmentally induced ASD pathophysiologyand should provide guidance for developing preventive and symptomatic treatments.Translational Psychiatry (2013) 3, e220; doi:10.1038/tp.2012.143; published online 22 January 2013
Introduction
Autism spectrum disorders (ASD) are a heterogeneous groupof neurodevelopmental disorders that are characterized byimpairments in social interaction and communication alongwith restrictive and repetitive behaviors.1 Many of thecognitive and behavioral features of ASD are believed toarise from central nervous system dysfunction, but abnorm-alities in many non-central nervous system tissues have beenassociated with ASD.2,3 Recent studies have implicated
abnormalities in systemic physiology that transcend organ
specific dysfunction, at least in some children with ASD.2–4
Thus, it is possible that organs other than the brain and/or
systemic abnormalities could be the source of the primary
pathophysiological that manifest, in part, with secondary brain
dysfunction.A recent meta-analysis found that 5% of children with ASD
meet criteria for a classic mitochondrial disease (MD) and
suggest that this subgroup has distinct clinical characteristics
1Department of Pediatrics, Arkansas Children’s Hospital Research Institute, Little Rock, AR, USA and 2The Kilee Patchell-Evans Autism Research Group-Departmentsof Psychology (Neuroscience) and Psychiatry, Lawson Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON,CanadaCorrespondence: Dr RE Frye, Department of Pediatrics, Arkansas Children’s Hospital Research Institute, Slot 512-41B, 13 Children0s Way, Little Rock, AR 72202, USA.E-mail [email protected] study has not been presented or published previously.
Received 2 July 2012; revised 27 October 2012; accepted 10 November 2012Keywords: acyl-carnitines; autism spectrum disorder; clostridia; microbiome; mitochondrial disease; propionic acid
Citation: Transl Psychiatry (2013) 3, e220; doi:10.1038/tp.2012.143& 2013 Macmillan Publishers Limited All rights reserved 2158-3188/13
that distinguish it from the general ASD population.3,5 Thisstudy also found that about 30% of the general ASDpopulation exhibited biomarkers consistent with MD.3 Thehigh prevalence of abnormal mitochondrial biomarkers in ASDhas been suggested to be due to mitochondrial dysfunctionthat is more prevalent and distinct from classic MD. Such anotion is supported by a recent study that found that 80% ofthe children with ASD demonstrated below normal function ofthe electron transport chain (ETC) in lymphocytes.6
The reason for mitochondrial dysfunction in ASD isunknown, but the fact that only 23% of children with ASDand MD have a known mitochondrial deoxyribonucleic acid(mtDNA) abnormality suggests that MD may be acquiredrather than genetic in many ASD cases.3 Indeed, some havesuggested that the systemic abnormalities in ASD such asmitochondrial dysfunction may arise from environmentaltriggers7 in genetically sensitive subpopulations.8,9 Entericshort-chain fatty-acids, such as propionic acid (PPA),10–17
which are fermentation by-products of ASD-associatedenteric bacteria (that is, Clostridia, Desulfovibrio, Sutterellaand Bacteroidetes), have been suggested as a possibleenvironmental triggers in ASD.18,19 Interestingly, humans withimpairments in PPA metabolism20–22 exhibit neurodevelop-mental conditions with ASD features.23
Recently, a rodent model has been development in whichreversible (30 min) bouts of ASD-type (that is, stereotyped,perseverative and impaired social) behaviors are produced bybrief intracerebroventricular infusions of PPA (http://www.psy-chology.uwo.ca/autism/autism6.htm for behavioral video).This animal model demonstrates several characteristics thathave been reported in ASD such as tics, electrographicseizures, innate neuroinflammation and redox, lipid, phos-phatidylethanolamine, mitochondrial, acyl-carnitine and car-nitine abnormalities.10–12,14–17 This animal model provides anunderstanding of how exogenous agents, such as PPA, cancause reversible behavioral, metabolic, neuropathologicaland neurophysiological changes associated with ASD. Mostimportantly, this animal model has predictive value as itdemonstrates biomarkers of abnormal mitochondrial fatty-acid metabolism (that is, acyl-carnitine elevations) that couldbe used as routine biomarkers if found in children with ASD.
Several lines of evidence suggest that mitochondrial fatty-acid oxidation could be abnormal in a subset of children withASD. First, free carnitine, the cofactor used to transport long-chain and very-long-chain fatty-acids into the mitochondrialmatrix, has been shown to be depleted in children with ASD.24
Free carnitine can be depleted if it remains bound tounprocessed fatty-acids due to a reduction in mitochondrialfatty-acid beta-oxidation.25 Second, elevations in long-chainand very-long-chain fatty-acids have been reported in childrenwith ASD as compared with controls, suggesting excessunprocessed fatty-acid in the serum of children with ASD.26
Third, a case study and case series of patients with ASD havereported elevations in acyl-carnitines, the standard biomarkerfor mitochondrial fatty-acid oxidation deficits.27,28 Thus, thereis ample evidence to suggest that abnormalities in fatty-acidmetabolism (that is, acyl-carnitine elevations) may be found inchildren with ASD.
Figure 1 demonstrates the acyl-carnitine elevations in brainhomogenates found in rats exposed to intracerebroventricular
infusions of PPA as compared with those exposed tophosphate buffered saline vehicle control. These abnormal-ities included short-chain (2–5 carbon length) and long-chain(13–18 carbon length) acyl-carnitines but not medium-chain(6–12 carbon length) acyl-carnitines.13 We hypothesize that asubset of children with ASD manifest biomarkers of abnormalmitochondrial fatty-acid metabolism that are similar to thosereported in the PPA rodent model of ASD. Here we review thecharts of consecutive patients seen in a medically basedautism clinic who underwent a systematic workup for mito-chondrial disorders per recently published guidelines, whichincluded screening for fatty-acid metabolism disorders.3
Overall, 17% of children with ASD were found to demonstratea unique pattern of acyl-carnitine abnormalities that weresimilar to the acyl-carnitine abnormalities found in the rodentPPA model of ASD. The potential causes of these abnormalitiesand their possible relation to ASD pathogenesis is discussed.
Materials and methods
Subject population. Parents of patients seen from 2008–2011 in a medically based autism clinic were requested toconsent to allow their child’s medical information to beanonymously abstracted into a clinical database that con-tained medical history, physical examination findings and theresults of neurological and metabolic testing. Approximately98% of parents (326 total patients) signed the consent.
Figure 1 Acyl-carnitine elevations in the brain of rats treated intracerebroven-tricularly with propionic acid. Notice that the majority of fatty-acid elevations were inshort-chain (2–5 carbon length) and long-chain (13–18 carbon length) fatty-acids ascompared with the medium-chain (6–12 carbon length) fatty-acids. This is adaptedfrom Thomas et al.13 where it was presented as a table.
Metabolic evaluation. A standardized metabolic workup formitochondrial metabolism disorders was conducted on mostpatients.29,30 The algorithm for this evaluation is depicted inFigure 2. Initial testing included laboratory tests to identifyabnormalities in the respiratory chain, tricarboxylic-acid cycle(TCAC) and fatty-acid oxidation pathways. Abnormalitiesdetected in initial testing were confirmed with repeat testing.If abnormalities could not be replicated, laboratories testswere reconsidered during metabolic stress or illness if a highindex of suspicion remained for the patient.
An acyl-carnitine panel, which measures short-chain,medium-chain and long-chain acyl-carnitines, was used asthe primarily laboratory test to detect defects in the fatty-acidoxidation pathway. An acyl-carnitine panel was measuredat initial testing in 213 of the consented patients. Theacyl-carnitine panel was considered abnormal if three ormore acyl-carnitines were elevated in the panel. An abnormalacyl-carnitine panel was confirmed by repeat testing.
If acyl-carnitine abnormalities were confirmed, non-mito-chondrial disorders of fatty-acid metabolism were ruled-outbefore a MD workup was initiated. Disorders ruled-outincluded generalized hyperlipidemia, hypercholesterolemia,multiple carboxylase deficiencies (that is, biotinidase defi-ciency), zinc deficiency, abnormal copper metabolism andhypoglycemia. After such disorders were ruled-out, a MDworkup was pursued. The initial step in the MD workup wasexamination for mtDNA gene abnormalities by either atargeted analysis for common mutations and/or deletions
and/or sequencing of the entire mtDNA genome (BaylorMedical Genetics Laboratory, Houston, TX, USA).31,32
When a conclusive mtDNA abnormality could not beidentified, nuclear mitochondrial gene testing and/or amuscle and/or skin biopsy was recommended. Nuclearmitochondrial gene abnormalities were ruled-out using anoligonucleotide array with comparative genomic hybridizationanalysis that examines B180 nuclear genes involved inmitochondria function, including genes involved in fatty-acid oxidation, carnitine metabolism, mitochondrial biogen-esis, mtDNA maintenance, transcription and translation, andETC complex assembly (MitoMet, Baylor Medical GeneticsLaboratory).
In some patients, the quadricep muscle was biopsied andanalyzed with light and electron microscopy, as well as formtDNA content.33,34 In some patients, fibroblasts obtainedfrom a skin biopsy were cultured. ETC function was examinedon frozen muscle and cultured fibroblasts (Baylor MedicalGenetics Laboratory).35 Both uncorrected ETC function andETC function correcting for citrate synthase are presented.Fibroblasts were incubated with d3-palmitate and L-carnitinein duplicate for 72 h to determine function of the fatty-acidoxidation pathway (Baylor Institute of Metabolic Disease,Dallas, TX).36
Determination of acyl-carnitine abnormalities. To calcu-late the prevalence of having an abnormal acyl-carnitinepanel in the ASD sample, the prevalence of having an acyl-
Non-mitochondrialdisorder confirmed
Skin and muscle biopsy Histology (muscle) Electron microscopy (muscle)Electron Transport Chain (muscle and skin)Beta-oxidation studies (skin)mtDNA content (muscle)
Targeted genetic testingMitochondrial DNA sequencing
Figure 2 Algorithm for metabolic workup of autistic spectrum disease patients evaluated in the medically based autism clinic. Patients are screened with biomarkers ofabnormal mitochondrial function in the fasting state. Abnormalities are verified with repeat fasting biomarker testing. For patients with biomarkers for a fatty-acid oxidationdefect, other disorders of fatty-acid metabolism are ruled-out before further workup for a mitochondrial disorder. Patients with consistent biomarkers for mitochondrialdysfunction are first investigated for genetic causes of their mitochondrial disorder before considering a muscle and/or skin biopsy. mtDNA, mitochondrial deoxyribonucleicacid; RBC, red-blood cell.
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carnitine panel with the first laboratory test was multiplied bythe percent of patients confirmed to have an abnormal acyl-carnitine panel on repeat laboratory testing. This was done toaccount for the fact that some patients did not repeat theacyl-carnitine panel even though it was abnormal. Todetermine the specific acyl-carnitine species that wereconsistently elevated across the subgroup of patients withconsistently abnormal acyl-carnitine panels, we examinedthe first two acyl-carnitine panels measured for each patient.Values for each individual acyl-carnitine species (for eachpatient) were transformed to a percent of the upper limit ofnormal for the specific acyl-carnitine species. The mean ands.e. were then calculated for each acyl-carnitine species tosummarize the group data. Statistical significance wascalculated as the significance of the difference betweenthe upper limit of normal and the group mean for eachacyl-carnitine specific using a z-distribution derived from thegroup mean and s.e.
Measurement of glutathione metabolism. Glutathionemetabolism was evaluated in four participants. Thesepatients were compared with normative values establishedin a previous study on redox metabolism.37 These controlsincluded 42 healthy children ranged from 2–7 years of agewith no history of developmental delay or neurologicalsymptoms. Independent sample t-tests were used forcomparison. Fasting blood samples were collected intoethylenediaminetetraacetic acid vacutainer tubes and wereimmediately chilled on ice before centrifuging at 4000 g for10 min at 4 1C. To prevent metabolite inter-conversion theice-cold samples were centrifuged within 15 min of the bloodcollection and the plasma stored at � 80 1C until analysiswithin 2 weeks. Details of the methodology for high-pressureliquid chromatography with electrochemical detection andmetabolite quantitation have been previously described.38
Total and free-reduced glutathione, oxidized glutathione(GSSG) and the total-reduced glutathione/GSSG and free-reduced glutathione/GSSG ratios were measured.
Results
Prevalence and patterns of abnormal acyl-carnitines.Seventy-four (35%) of the 213 patients tested demonstratedan increase in three or more acyl-carnitines when initiallymeasured. Forty-two (57%) of the 74 underwent repeat acyl-carnitine testing. Three or more acyl-carnitines were abnor-mal a second time in 20 (48%) of the 42 patients, resulting ina prevalence of 17% of ASD children who manifestedconsistent acyl-carnitine panel abnormalities. Figure 3demonstrates the mean values of each acyl-carnitine speciesrelative of the upper limit of normal. C4OH, C14 and C16:1were significantly elevated as compared with the upper limitof normal (z¼ 2.18, P¼ 0.01; z¼ 5.71, Po0.0001; z¼ 2.85,P¼ 0.02, respectively), and were 186%, 226% and 131% ofthe upper limit of normal, respectively.
Clinical characteristics. Clinical characteristics of the 20patients with consistent elevations in the acyl-carnitine panelare given in Supplementary Table 1. The average age was8.7 years (s.d. 2.25) with a male to female ratio of 3:1.
Autistic disorder was diagnosed in 70% of the participantswhile 25% had a diagnosis of pervasive developmentaldisorder-not otherwise specified and 5% were diagnosedwith Asperger syndrome. Developmental regression wasreported in 45% of patients.
Glutathione metabolism. The subset of children in whichglutathione metabolism was examined demonstrated signifi-cantly lower total-reduced glutathione (t¼ 12.75, Po0.0001)and free-reduced glutathione (t¼ 10.04, Po0.0001) valuesand total-reduced glutathione/GSSG (t¼ 9.07, Po0.001) andfree-reduced glutathione/GSSG (t¼ 4.69, Po0.0001) ratios aswell as higher GSSG (t¼ 2.61, P¼ 0.01) values as comparedwith typically developing controls (Figure 4), suggesting both areduction in the production of gluthathione and increase ingluthathione utilization by reactive oxygen species.
Genetic characteristics. Nuclear DNA examinations werenormal in the great majority of patient (94%) in which suchexaminations were conducted. mtDNA was normal in 85% ofthe patients in which it was examined. The two mtDNAabnormalities that were identified involved novel maternallyinherited homoplasmic cytochrome B gene mutations
Figure 3 Average acyl-carnitine values (with s.e. bars) from 20 patients withconsistent abnormal elevations in multiple acyl-carnitines. Acyl-carnitine values arerepresented as percent upper limit of normal for each acyl-carnitine species. Noticethat C4OH, C14 and C16:1 are significantly elevated as compared with themaximum upper limit of normal.
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(15533A4G and 15404T4C), which altered evolutionaryconserved amino acids. mtDNA content in muscle from fourpatients ranged from 109–189% of normal with a mean of160.5% (s.d.±27%).
Neurological and biochemical testing. Neurological andbiochemical characteristics of the patients are given inSupplementary Table 2. No abnormalities were found inthe majority of patients that underwent an extended 23 hvideo electroencephalogram. Acyl-glycine panel, aminoacids, glucose, insulin, Co-Q10, biotin, cholesterol andtriglyceride levels were unremarkable in all patients in whichthey were tested. Urine organic acids were abnormal in themajority in which it was tested with elevations in TCACmetabolites, specifically elevations in citrate and/or isocitraterepresenting the majority of the abnormalities. Lactate waselevated in about half of the patients. Carnitine panelwas abnormal in half of the patients in whom it was measuredwith 33% of the patients having high-esterified carnitine and17% having low free carnitine. Creatine kinase and pyruvatewas elevated in a minority of patients in which it wasmeasured. Interestingly, red-blood cell zinc was borderlinelow in 70% of the patients in which it was measured and red-blood cell copper was slightly elevated in 35% of the patients
in which it was measured. The great majority of individuals(90%) in which red-blood cell zinc and red-blood cellcopper were both measured demonstrated an abnormalityin at least one.
Muscle histology. All five of the patients that underwentmuscle biopsy demonstrated abnormal histological andelectron microscopy findings. Four demonstrated fibertype 1 predominance with two also demonstrating fiber type2 atrophy. The fifth demonstrated myofiber size irregularity,increased sarcoplasmic lipid and scattered succinate dehy-drogenase hyper-reactive fibers. Electron microscopydemonstrated an increased number of mitochondria in thesubsarcolemmal region in all and also in the intermyofibrillarregion in two. Mitochondria were maloriented in two patientsand degeneration of membranous organelles was seen inthree cases.
ETC and fatty-acid oxidation function. ETC functionwas testing on all five muscles biopsies. Corrected anduncorrected ETC activity is shown in Figures 5a and b,respectively, and demonstrate a partial defect in complexesI/III and I/III rotenone sensitive (RS). ETC and fatty-acidoxidation testing was conducted on fibroblast cultures from
Figure 4 Gluthathione abnormalities in four children with consistent elevations in multiple acyl-carnitine species. Notice that the patients have lower total (tGSH, mM) andfree (fGSH, mM) reduced gluthathione, as well as lower tGSH/fGSSG (free-oxidized gluthathione, mM) and fGSH/fGSSG ratios and higher fGSSG as compared with typicallydeveloping controls, suggesting both a reduction in the production of gluthathione and increase in gluthathione utilization by reactive species.
Figure 5 Electron transport chain (ETC) function of muscle (a, b) and fibroblast culture (c, d), as well as function of the fatty-acid oxidation pathway in fibroblast cultures(e, f). Graph values represent percent of normal ETC function, uncorrected (a, c) or corrected for citrate synthase (b, d). Muscle ETC results suggest a partial defectin complexes I/III and I/III rotenone sensitive (RS) while fibroblast culture ETC function suggests a partial defect in complex II/III activity. In fibroblast culture ETC studiescomplexes I/III RS and IV demonstrate considerable variability due to overactivity (4200% of the mean) in complex I/III RS in three patients and complex IV in onecase. Fatty-acid oxidation values represent mean of specific acyl-carnitine species (higher is worse) uncorrected (e) and corrected for citrate synthase (f). Elevation in theshort-chain fatty-acid D3-C4 was due to three patients demonstrating high D4-C4 values. The one patient with a significantly elevated D4-C4 value was found not to have amutation in ;the short-chain acyl-CoA dehydrogenase gene suggesting that the abnormalities in fatty-acids in fibroblast culture were due to other mitochondrial metabolismabnormalities.
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eight patients. Overall complex II/III activity was deficientacross patients, and complex I/III RS demonstrated variableand elevated (4200% of the mean) activity, which was alsoseen to a lesser extent in complex IV (Figures 5c and d).Functional fatty-acid oxidation testing demonstrated eleva-tions in the short-chain fatty-acid D3-C4 (Figures 5e and f).
This effect was due to three patients with high D4-C4. Onlyone patient had a high enough elevation for a short-chainacyl-CoA dehydrogenase defect to be considered. However,sequencing of exons 1–10 of the short-chain acyl-CoAdehydrogenase gene (GeneDx, Gaithersburg, MD, USA)39
for the patient was normal.
ElectronTransport
Chain
Fatty Acids
Fatty Acids
Pyruvate
Acetyl-CoA
Acetyl-CoA
Complex INADH
CitrateOxalacetate NADH
Complex II
Complex III
IsocitrateTricarboxylicAcid Cycle
TricarboxylicAcid Cycle
Malate
NADH
Fumarate α-ketoglutarate
α-ketoglutarate
Complex IV
Succinyl-CoA
Succinyl-CoA
SuccinateComplex V
FADH2
FADH2
PyruvateLactate
NADHNADH
CitrateOxalacetate
Distal Malate ProximalIsocitrate
Fumarate NADH
Succinate
Methylmalonyl-CoAPropionic Acid Propionyl-CoA
Lactate
a
b
Figure 6 The tricarboxylic-acid cycle during (a) typical metabolism and (b) with high levels of propionic acid. Propionic acid is metabolized to propionyl-CoA, which inhibitsthe proximal portion of the tricarboxylic-acid cycle and enhances the distal portion of the tricarboxylic-acid cycle (see discussion for details). FADH2, flavin adenine dinucleotide;NADH, nicotinamide adenine dinucleotide.
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MD diagnosis criteria. Using the Morava et al.40 criteria forthe five patients that underwent muscle biopsy found thatthree patients were rated as having a definite MD and twopatients were rated as having probable MD.
Discussion
In this report, 17% of a large cohort of children with ASDdemonstrated consistent elevations in short-chain and long-chain, but not medium-chain, acyl-carnitines. This pattern ofacyl-carnitine abnormalities is similar to elevations in brainacyl-carnitines seen in the PPA rodent model of ASD.13 Othermetabolic abnormalities, specifically mitochondrial dysfunc-tion and glutathione abnormalities, were identified in thepatient cohort that are similar to the PPA rodent model of ASD.Such abnormalities are discussed below in detail.
Mitochondrial abnormalities is ASD patients are consis-tent with PPA toxicity. ETC function testing in muscledemonstrated a partial deficit in complexes I/III and I/III RSactivity. These ETC abnormalities, along with other biomar-kers of mitochondrial dysfunction, are consistent with PPAinterfering with mitochondrial metabolism, potentially throughthe TCAC. The TCAC utilizes two electron carriers,nicotinamide adenine dinucleotide (NADH) and flavin ade-nine dinucleotide (FADH2) to shuttle electrons to complexes Iand II of the ETC, respectively. Normally TCAC reactionsproduce 3 NADH and 1 FADH2, resulting in a 3:1 NADH toFADH2 ratio (Figure 6a).
PPA is metabolized to propionyl-CoA, which is furthermetabolized to produce methymalonic-CoA. Methymalonic-CoA enters the TCAC half way through the cycle as succinyl-CoA, thereby essentially ‘short circuiting’ the TCAC(Figure 6b). Elevated succinyl-CoA enhances the distal halfof the TCAC and inhibits the proximal half of the TCAC. As thedistal half of the TCAC produces 1 NADH and 1 FADH2, if theproximal half of the TCAC is inhibited, the NADH to FADH2
ratio will change from 3:1 to 1:1. As NADH is metabolized bycomplex I a reduction in the production in NADH will result in arelative deficit in complex I, consistent with the findings fromthe ETC muscle studies. In addition, inhibition of the proximalportion of the TCAC will also result in a build-up of the firstmetabolites in the TCAC, consistent with the elevations incitrate and isocitrate in our patients. Furthermore, the endproduct of the fatty-acid oxidation pathway, acetyl-CoA, is thefirst metabolite of the proximal half of the TCAC. Thus,inhibition of the proximal half of the TCAC inhibits the fatty-acid oxidation pathway. This is consistent with the fact thatexamination of the fatty-acid oxidation pathway in fibroblastculture did not reveal any abnormalities to explain theelevations in acyl-carnitines.
Examination of fibroblasts, which occurs after 6 or moreweeks of fibroblast growth in culture, demonstrated aboveaverage activity of complexes I/III RS and IV and a partialdeficit in complex II/III. Interestingly, overactivity in complexesI and IV have been reported in children with ASD/MD.41,42 Thedisparity in the ETC findings between muscle and fibroblastculture can be explained by the alternative use of citratesynthase, the first enzyme in the TCAC, for metabolizingpropionyl-CoA. Normally citrate synthase produces citrate
from acetyl-CoA and oxaloacetate. In the context of highlevels of propionyl-CoA, citrate synthase produces methylcitrate, a dead end metabolite, from propionyl-CoA. This willalso result in a competition for citrate synthase by bothpropionyl-CoA and acetyl-CoA, further blocking the metabo-lism of metabolic pathways that produce acetyl-CoA as an endproduct, such as the fatty-acid oxidation pathway. Thisoveruse of citrate synthase will most likely also result in anupregulation of citrate synthase over time.
If an agent, such as PPA, that suppressed mitochondrialfunction was present in vivo but not in vitro, mitochondrialfunction in the muscle, but not the fibroblast culture, would bemore compromised. If upregulation of citrate synthaseoccurred due to excess PPA in vivo and then PPA wasremoved in vitro, the high activity of citrate synthase in vitrowould overproduce citrate and enhance the proximal portion ofthe TCAC, which preferentially produces NADH, the electroncarrier metabolized by complex I. This is consistent with theobserved I/III RS overactivity seen in fibroblast culture.
One common theme of the observed ETC dysfunction inboth muscle and fibroblast culture was that complex dysfunc-tion primarily occurred when evaluating the function ofcomplex III with complex I or II, suggesting that it is theinteraction between complex III and complex I or II rather thanat complex I or II specifically. Interesting, the 15533A4Gcytochrome b mutation identified in one of the cases has beenshown to have a complicated effect on complex III function.Rather than causing a frank decrease in complex III function,this mutation appears to result in delayed assembly of theI,III,IV supercomplex,43 thus influencing the interaction ofcomplex III with other complexes rather than specificallyaffecting only complex III. Interestingly, PPA has been shownto have its detrimental effect on the ETC through inhibition ofcomplex III function.44 Furthermore, alterations in brainomega 3/6 cardiolipin profiles found in the PPA rodent modelcould change inner mitochondrial membrane fluidity, and,thus, could potentially affect mitochondrial ETC complexinteractions.13
Mitochondrial abnormalities are consistent withacquired MD. All patients that underwent a workup for MDdemonstrated probable or definite MD by standardizedcriteria.2,40 However, the majority of the patients in this studydid not have any identifiable genetic causes for their MD.This is not surprising as only 23% of children with ASD andMD have a known mtDNA abnormality.3 The effect of thecytochrome b mutation found in two boys is complicated.45,46
For example, delayed supercomplex assembly associatedwith the 15533A4G gene mutation in a child with aneurodevelopment disorder was restored when mutanttransmitochondrial cybrids were developed from the15533A4G case.43 This suggests that this mtDNA mutationis a risk factor that requires interactions with nuclearmutations, polymorphisms or epigenetics and/or environ-mental triggers or modulators in order for the diseasephenotype to be expressed.43 Thus, the characteristics ofthis series of patient are consistent with the notion that thesystemic abnormalities seen in this subgroup of ASD patientsmay arise from environmental triggers7 in geneticallysensitive subpopulations.8,17,47,48
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Glutathione metabolism and oxidative stress abnormal-ities in ASD patients. Four patients in the series underwentmeasurements of glutathione metabolism. Overall, there wasa marked decreased in total and free-reduced glutathionewith a slight increase in GSSG, suggesting a primary deficitin the production of glutathione and increase in utilization.This finding parallels the PPA rodent animal model. Indeed,intracerebroventricular infusion of PPA in rats decreasestotal glutathione in brain homogenates.11 Further evidencefor increased oxidative stress has been demonstrated in therodent PPA model, including increased brain protein carbo-nylation and lipid peroxidation, altered phospholipid profilesand increased activated microglia.11,13,16 Such findings areall consistent with those from ASD patients.49–51
Abnormalities in zinc and copper in ASD patients. Inter-estingly, a large proportion of the patients in the seriesdemonstrated mild abnormalities in zinc and/or copperconcentrations. Such abnormalities have been reported inthe ASD population previously52–54 and zinc supplementa-tion (along with B6) has been shown to decrease copperlevels and improve function in ASD in an uncontrolledstudy.54 Some have hypothesized that abnormalities in zincand copper metabolism could result in poor metallothioneinfunction, leading to susceptibility to environmental toxicantsthrough increased oxidative stress or mitochondrial dysfunc-tion.52 Interestingly, low zinc levels have been associatedwith pediatric inflammatory bowel disease55 and increasedinflammation in animal models of colitis56 and zinc supple-mentation appears to be protective of bowel inflammation inclinical57 and animal studies.58 Although speculative, thismay occur via a decrease in the activity of coloprotectivemetallothionein59 in the intestinal mucosa, or impairment ofT- and B-cell interaction60 that may contribute to gutdysbiosis favoring ASD-associated bacteria.
Potential links to unique ASD microbial populations.Enteric bacterial populations found in increased numbers instool samples of ASD patients (Clostridia, Desulfovibrio) areknown to produce PPA from fermentation of dietarycarbohydrates.18,19,31 Impaired carbohydrate digestion andtransport in children with ASD can result in a higherconcentration of dietary carbohydrates for these bacterialpopulations to ferment.18,19,61 A recent study has shown thatstool from ASD patients have elevations in PPA and othershort-chain fatty-acids.62 In addition, Desulfovibrio is capableof producing PPA from fermentation of peptones and canproduce hydrogen sulfide, a potential mitochondrial toxin,which may act synergistically with PPA to promote mitochon-drial dysfunction.17,18,63,64
Interestingly, administration of common antibiotics (that is,beta lactams) for routine pediatric infections alters gut florafavoring PPA-producing species. This could be significantconsidering the reported high incidence of antibiotic use insome ASD patients.18,19,65,66 In addition, this offers a potentialexplanation for temporary behavioral improvements in somepatients following vancomycin or metronidazole treatment,which eradicates these bacteria18,19,66 and profoundlyreduces stool PPA.67 Furthermore, removal of refinedcarbohydrates from the diet, which has been suggested as
an empiric treatment to improve the behavioral fluctuations,gastrointestinal symptoms and dysbiosis in ASD,8 may act byreducing substrate for these bacteria to produce PPA.11
Although low concentrations of PPA may be beneficial,humans with impairments in PPA metabolism (that is,propionic or methymalonic acidemia, holocarboxylase, bioti-nidase or B12 deficiency, valproate or ethanol exposure)exhibit neurodevelopmental conditions with behavioral andbiochemical similarities to ASD.68,69 PPA and related short-chain fatty-acids (that is, butyrate and acetate) have broadeffects on cellular systems.13,20,26,27,48,70–72 They are activelytaken up into the brain73 and can affect diverse physiologicalprocesses such as cell signaling,71 neurotransmitter synth-esis and release,70 mitochondrial function,20 lipid metabo-lism,13,74 immune function,75 cell–cell interactions76 and geneexpression.72 Thus, there are many potential mechanismswhere metabolic end products of the enteric microbiome canalter host physiology.17
Of particular interest, PPA is a known inhibitor of mitochon-drial function, through sequestration of carnitine and theproduction of propionyl-CoA, a potential cytotoxin.44,77 Methy-malonic acid, a metabolite of PPA, results in abnormalmitochondrial morphology,78 tissue specific ETC dysfunction,79
inhibition of the complex I and II function when interacting withcomplex III in the brain80 and reductions in reduced glu-tathione,78 similar to the patient cohort presented and the PPAanimal model. As PPA is metabolized through the TCAC, wepropose that excess exogenous PPA or related short-chainfatty-acid metabolites interfere with mitochondrial metabolism,thereby causing acquired mitochondrial dysfunction.
Impaired carnitine metabolism can act synergisticallywith PPA-producing bacteria. The Naþ dependentorganic cation/carnitine transporter 2 transports carnitineacross the gut-blood and blood-brain barriers.81 Antibiotics(that is, beta lactams) commonly used to treat pediatric,infections directly inhibit the organic cation/carnitine trans-porter 2 transporter, thus directly impairing carnitine reab-sorption.81 This could be significant considering the highincidence of antibiotic use in ASD patients, which can alsopromote gut dysbiosis favoring ASD-associated gut bacterialpopulations that produce PPA.18,19,65,66 Given that bothcarnitine deficiency and PPA can be detrimental to mito-chondrial metabolism, it is possible that antibiotic overusecan cause these two effects to act synergistically to cause anacquired mitochondrial disorder, especially in geneticallysusceptible individuals.
Interestingly, children with ASD, as a group, have beenfound to have reduced blood carnitine3 and a X-linked inbornerror of carnitine biosynthesis has been shown to be a riskfactor for ASD.82 Furthermore, oral carnitine, and its derivativeacetyl-L-carnitine, have both neuroprotective83,84 and colo-protective properties.85 Given that carnitine supplementationimproves function in children with ASD,86,87 it deserves furtherinvestigation as a therapeutic agent in ASD.3,86,87
Summary. This study has demonstrated that B17% ofchildren with ASD manifest biomarkers of abnormal mito-chondrial fatty-acid metabolism that parallel similar biomar-kers in the PPA rodent model of ASD. Detailed examination
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of a subset of these patients indicates that these metabolicabnormalities are at least partly due to TCAC and ETCdysfunction. Genetic disorders do not appear to account forthe majority of these cases and the two individuals withabnormalities in mtDNA suggest dysfunction in the inter-action of complex III with complex I and/or II. For the cases inwhich genetic abnormalities have not been found it is verylikely that MD is acquired. As this subgroup of ASD patientshave several parallels with the rodent PPA model of ASD,13
this rodent model may be a useful tool to further examine thetemporal relation of behavioral bouts in relation to carnitine-acyl-carnitine fluctuations, and their possible response totherapeutic compounds thought to be useful in the treatmentof ASD and mitochondrial dysfunction such as carnitinesupplementation.
It is important to note that PPA affects multiple systems in acomplex manner and the evidence of increased PPA or othershort-chain fatty-acids being involved in the pathophysiologyof ASD, although compelling, is circumstantial at this stage.Thus, future studies should identify additional parallelsbetween the PPA rodent model of ASD and individuals withASD who manifest similar biomarkers. Further study of thismodel and this subgroup of ASD patients should improve ourunderstanding of the pathophysiology and potential riskfactors that lead to the metabolic, brain and behaviorabnormalities associated with ASD.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements. This research was supported in part from generouscontributions from GoodLife’s Children’s Foundation and Autism Research Instituteto DFM. We thank Dr Raymond Thomas (postdoctoral fellow) for published data onbrain lipids and Drs Stephan Kahler and Anirudh Saronwala for their insight intopropionic metabolism.
1. APA Diagnostic and statistical manual of mental disorders. 4th edn DC: AmericanPsychiatric Association: Washington, 1994.
2. Frye RE, Rossignol DA. Mitochondrial dysfunction can connect the diversemedical symptoms associated with autism spectrum disorders. Pediatr Res 2011; 69(5Pt 2): 41R–47RR.
3. Rossignol D, Frye RE. Mitochondrial dysfunction in autism spectrum disorders: Asystematic review and meta-analysis. Mol Psychiatry 2012; 17: 290–314.
4. Rossignol DA, Frye RE. A review of research trends in physiological abnormalities inautism spectrum disorders: immune dysregulation, inflammation, oxidative stress,mitochondrial dysfunction and environmental toxicant exposures. Mol Psychiatry 2012;17: 389–401.
5. Weissman JR, Kelley RI, Bauman ML, Cohen BH, Murray KF, Mitchell RL et al.Mitochondrial disease in autism spectrum disorder patients: a cohort analysis. PLoS ONE2008; 3: e3815.
6. Giulivi C, Zhang YF, Omanska-Klusek A, Ross-Inta C, Wong S, Hertz-Picciotto I et al.Mitochondrial dysfunction in autism. JAMA 2010; 304: 2389–2396.
7. Hallmayer J, Cleveland S, Torres A, Phillips J, Cohen B, Torigoe T et al. Genetic heritabilityand shared environmental factors among twin pairs with autism. Arch Gen Psychiatry2011; 68: 1095–1102.
8. Herbert MR, Russo JP, Yang S, Roohi J, Blaxill M, Kahler SG et al. Autism andenvironmental genomics. Neurotoxicology 2006; 27: 671–684.
9. Ashwood P, Anthony A, Torrente F, Wakefield AJ. Spontaneous mucosal lymphocytecytokine profiles in children with autism and gastrointestinal symptoms: mucosal immuneactivation and reduced counter regulatory interleukin-10. J Clin Immunol 2004; 24: 664 73.
10. MacFabe DF, Rodrı́guez-Capote K, Hoffman JE. A novel rodent model of autism:intraventricular infusions of propionic acid increase locomotor activity and induceneuroinflammation and oxidative stress in discrete regions of adult rat brain. Am J BiochemBiotechnol 2008; 4: 146–166.
11. MacFabe DF, Cain DP, Rodriguez-Capote K, Franklin AE, Hoffman JE, Boon F et al.
Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain
fatty acids on the pathogenesis and characteristics of autism spectrum disorders. Behav
Brain Res 2007; 176: 149–169.12. Shultz SR, MacFabe DF, Ossenkopp KP, Scratch S, Whelan J, Taylor R et al.
Intracerebroventricular injection of propionic acid, an enteric bacterial metabolic end-
product, impairs social behavior in the rat: implications for an animal model of autism.
Neuropharmacology 2008; 54: 901–911.13. Thomas RH, Foley KA, Mepham JR, Tichenoff LJ, Possmayer F, MacFabe DF. Altered
brain phospholipid and acylcarnitine profiles in propionic acid infused rodents: further
development of a potential model of autism spectrum disorders. J Neurochem 2010; 113:
515–529.14. MacFabe DF, Cain NE, Boon F, Ossenkopp KP, Cain DP. Effects of the enteric bacterial
metabolic product propionic acid on object-directed behavior, social behavior, cognition,
and neuroinflammation in adolescent rats: relevance to autism spectrum disorder. Behav
Brain Res 2011; 217: 47–54.15. Shultz SR, Macfabe DF, Martin S, Jackson J, Taylor R, Boon F et al. Intracerebroven-
tricular injections of the enteric bacterial metabolic product propionic acid impair cognition
and sensorimotor ability in the Long-Evans rat: further development of a rodent model of
autism. Behav Brain Res 2009; 200: 33–41.16. Thomas RH, Meeking MM, Mepham JR, Tichenoff L, Possmayer F, Liu S et al. The enteric
further development of a rodent model of autism spectrum disorders. J Neuroinflammation
2012; 9: 153.17. MacFabe DF. Short-chain fatty acid fermentation products of the gut microbiome:
implications in autism spectrum disorders. Microb Ecol Health Dis 2012; 23: 19260.18. Finegold SM, Dowd SE, Gontcharova V, Liu C, Henley KE, Wolcott RD et al.
Pyrosequencing study of fecal microflora of autistic and control children. Anaerobe 2010;
16: 444–453.19. Finegold SM, Molitoris D, Song Y, Liu C, Vaisanen ML, Bolte E et al. Gastrointestinal
microflora studies in late-onset autism. Clin infect Dis 2002; 35(Suppl 1): S6–S16.20. Wajner M, Latini A, Wyse AT, Dutra-Filho CS. The role of oxidative damage in the
neuropathology of organic acidurias: insights from animal studies. J Inherit Metab Dis 2004;
27: 427–448.21. Coulter DL. Carnitine, valproate, and toxicity. J Child Neurol 1991; 6: 7–14.22. Calabrese V, Rizza V. Formation of propionate after short-term ethanol treatment and its
interaction with the carnitine pool in rat. Alcohol 1999; 19: 169–176.23. Al-Owain M, Kaya N, Al-Shamrani H, Al-Bakheet A, Qari A, Al-Muaigl S et al. Autism
spectrum disorder in a child with propionic acidemia. JIMD Reports 2012; 7: 63–66.24. Mostafa GA, El-Gamal HA, El-Wakkad ASE, El-Shorbagy OE, Hamza MM. Polyunsatu-
rated fatty acids, carnitine and lactate as biological markers of brain energy in autistic
children. Int J Child Neuropsychiatry 2005; 2: 179–188.25. Haas RH, Parikh S, Falk MJ, Saneto RP, Wolf NI, Darin N et al. The in-depth evaluation of
suspected mitochondrial disease. Mol Genet Metab 2008; 94: 16–37.26. Pastural E, Ritchie S, Lu Y, Jin W, Kavianpour A, Khine Su-Myat K et al. Novel plasma
phospholipid biomarkers of autism: mitochondrial dysfunction as a putative causative
mechanism. Prostaglandins Leukot Essent Fatty Acids 2009; 81: 253–264.27. Clark-Taylor T, Clark-Taylor BE. Is autism a disorder of fatty acid metabolism? possible
dysfunction of mitochondrial beta-oxidation by long chain acyl-CoA dehydrogenase. Med
Hypotheses 2004; 62: 970–975.28. Frye RE. Biomarkers of abnormal energy metabolism in children with autism spectrum
disorder. NAJ Med Sci 2012; 5: 141–147.29. Munnich A, Rustin P. Clinical spectrum and diagnosis of mitochondrial disorders. Am J Med
Genet 2001; 106: 4–17.30. Mitochondrial Medicine Society’s Committee on DHaas RH, Parikh S, Falk MJ, Saneto RP,
Wolf NI, Darin N et al. The in-depth evaluation of suspected mitochondrial disease.
Mol Genet Metab 2008; 94: 16–37.31. Wong LJ. Pathogenic mitochondrial DNA mutations in protein-coding genes. Muscle Nerve
2007; 36: 279–293.32. Wong LJ, Cobb BR, Chen TJ. Molecular analysis of mitochondrial DNA point mutations by
polymerase chain reaction. Methods Mol Biol 2006; 336: 135–143.33. Bai RK, Perng CL, Hsu CH, Wong LJ. Quantitative PCR analysis of mitochondrial DNA
content in patients with mitochondrial disease. Ann N Y Acad Sci 2004; 1011: 304–309.34. Bai RK, Wong LJ. Simultaneous detection and quantification of mitochondrial DNA
deletion(s), depletion, and over-replication in patients with mitochondrial disease. J Mol
Diagn 2005; 7: 613–622.35. Kirby DM, Thorburn DR, Turnbull DM, Taylor RW. Biochemical assays of respiratory chain
complex activity. Methods Cell Biol 2007; 80: 93–119.36. Roe CR, Roe DS. Recent developments in the investigation of inherited metabolic
disorders using cultured human cells. Mol Genet Metab 1999; 68: 243–257.37. James SJ, Melnyk S, Fuchs G, Reid T, Jernigan S, Pavliv O et al. Efficacy of
methylcobalamin and folinic acid treatment on glutathione redox status in children with
autism. Am J Clin Nutr 2009; 89: 425–430.38. Melnyk S, Pogribna M, Pogribny I, Hine RJ, James SJ. A new HPLC method for the
simultaneous determination of oxidized and reduced plasma aminothiols using coulometric
39. Corydon MJ, Vockley J, Rinaldo P, Rhead WJ, Kjeldsen M, Winter V et al. Role of common
gene variations in the molecular pathogenesis of short-chain acyl-CoA dehydrogenasedeficiency. Pediatr Res 2001; 49: 18–23.
40. Morava E, van den Heuvel L, Hol F, de Vries MC, Hogeveen M, Rodenburg RJ et al.Mitochondrial disease criteria: diagnostic applications in children. Neurology 2006; 67:
1823–1826.41. Graf WD, Marin-Garcia J, Gao HG, Pizzo S, Naviaux RK, Markusic D et al. Autism
associated with the mitochondrial DNA G8363A transfer RNA(Lys) mutation. J Child Neurol
2000; 15: 357–361.42. Frye RE, Naviaux RK. Autistic disorder with complex IV overactivity: a new mitochondrial
syndrome. J Ped Neurol 2011; 9: 427–434.43. Gil Borlado MC, Moreno Lastres D, Gonzalez Hoyuela M, Moran M, Blazquez A, Pello R et
al. Impact of the mitochondrial genetic background in complex III deficiency. PloS one
2010; 5: e12801.44. Schwab MA, Sauer SW, Okun JG, Nijtmans LG, Rodenburg RJ, van den Heuvel LP et al.
Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for
endogenous mitochondrial toxins. Biochem J 2006; 398: 107–112.45. Gil Borlado MC, Moreno Lastres D, Gonzalez Hoyuela M, Moran M, Blazquez A, Pello R et
al. Impact of the mitochondrial genetic background in complex III deficiency. PLoS ONE2010; 5: e12801.
46. Frye RE. Novel cytochrome b gene mutations causing mitochondrial disease in autism.
J Ped Neurol 2012; 10: 1–6.47. Ashwood P, Van de Water J. Is autism an autoimmune disease? autoimmunity reviews
2004; 3: 557–562.48. Ashwood P, Van de Water J. A review of autism and the immune response. Clin Dev
Immunol 2004; 11: 165–174.49. James SJ, Melnyk S, Jernigan S, Cleves MA, Halsted CH, Wong DH et al. Metabolic
endophenotype and related genotypes are associated with oxidative stress in children with
autism. Am J Med Genet B, Neuropsychiatr Genet 2006; 141B: 947–956.50. Chauhan A, Chauhan V. Oxidative stress in autism. Pathophysiology 2006; 13: 171–181.51. Wegiel J, Kuchna I, Nowicki K, Imaki H, Wegiel J, Marchi E et al. The neuropathology of
autism: defects of neurogenesis and neuronal migration, and dysplastic changes. Acta
Neuropathol 2010; 119: 755–770.52. Faber S, Zinn GM, Kern JC 2nd, Kingston HM. The plasma zinc/serum copper ratio as a
biomarker in children with autism spectrum disorders. Biomarkers 2009; 14: 171–180.53. Yasuda H, Yoshida K, Yasuda Y, Tsutsui T. Infantile zinc deficiency: association with
autism spectrum disorders. Scientific reports 2011; 1: 129.54. Russo AJ, Devito R. Analysis of copper and zinc plasma concentration and the
efficacy of zinc therapy in individuals with asperger’s syndrome, pervasive developmental
disorder not otherwise specified (PDD-NOS) and autism. Biomark Insights 2011; 6:
127–133.55. Sikora SK, Spady D, Prosser C, El-Matary W. Trace elements and vitamins at diagnosis in
pediatric-onset inflammatory bowel disease. Clin Pediatr 2011; 50: 488–492.56. Iwaya H, Kashiwaya M, Shinoki A, Lee JS, Hayashi K, Hara H et al. Marginal zinc
deficiency exacerbates experimental colitis induced by dextran sulfate sodium in rats.J Nutr 2011; 141: 1077–1082.
57. Scrimgeour AG, Condlin ML. Zinc and micronutrient combinations to combat
gastrointestinal inflammation. Curr Opin Clin Nutr Metab Care 2009; 12: 653–660.58. Barollo M, Medici V, D’Inca R, Banerjee A, Ingravallo G, Scarpa M et al. Antioxidative
potential of a combined therapy of anti TNFalpha and Zn acetate in experimental colitis.World J Gastroenterol 2011; 17: 4099–4103.
59. Tran CD, Ball JM, Sundar S, Coyle P, Howarth GS. The role of zinc and metallothionein inthe dextran sulfate sodium-induced colitis mouse model. Dig Dis Sci 2007; 52: 2113–2121.
60. Zhang Y, Okamura S, Kudo T, Masuo T, Mori M. Calcineurin inhibition by polaprezinc in
rats with experimentally-induced colitis. Life Sci 2011; 88: 432–439.61. Williams BL, Hornig M, Buie T, Bauman ML, Cho Paik M, Wick I et al. Impaired
carbohydrate digestion and transport and mucosal dysbiosis in the intestines of childrenwith autism and gastrointestinal disturbances. PLoS One 2011; 6: e24585.
62. Wang L, Christophersen CT, Sorich MJ, Gerber JP, Angley MT, Conlon MA. Elevated fecalshort chain fatty acid and ammonia concentrations in children with autism spectrum
disorder. Dig Dis Sci 2012; 57: 2096–2102.63. Barton LL, Fauque GD. Biochemistry, physiology and biotechnology of sulfate-reducing
bacteria. Adv Appl Microbiol 2009; 68: 41–98.64. Lagoutte E, Mimoun S, Andriamihaja M, Chaumontet C, Blachier F, Bouillaud F. Oxidation
of hydrogen sulfide remains a priority in mammalian cells and causes reverse electron
transfer in colonocytes. Biochim Biophys Acta 2010; 1797: 1500–1511.65. Fallon J. Could one of the most widely prescribed antibiotics amoxicillin/clavulanate
"augmentin" be a risk factor for autism? Med Hypotheses 2005; 64: 312–315.
66. Sandler RH, Finegold SM, Bolte ER, Buchanan CP, Maxwell AP, Vaisanen ML et al. Short-term benefit from oral vancomycin treatment of regressive-onset autism. J Child Neurol2000; 15: 429–435.
67. Mellon AF, Deshpande SA, Mathers JC, Bartlett K. Effect of oral antibiotics on intestinalproduction of propionic acid. Arch Dis Child 2000; 82: 169–172.
68. Yorifuji T, Kawai M, Muroi J, Mamada M, Kurokawa K, Shigematsu Y et al. Unexpectedlyhigh prevalence of the mild form of propionic acidemia in Japan: presence of a commonmutation and possible clinical implications. Hum Genet 2002; 111: 161–165.
69. Perez B, Desviat LR, Rodriguez-Pombo P, Clavero S, Navarrete R, Perez-Cerda C et al.Propionic acidemia: identification of twenty-four novel mutations in Europe and NorthAmerica. Mol Genet Metab 2003; 78: 59–67.
70. DeCastro M, Nankova BB, Shah P, Patel P, Mally PV, Mishra R et al. Short chain fattyacids regulate tyrosine hydroxylase gene expression through a cAMP-dependent signalingpathway. Brain Res Mol Brain Res 2005; 142: 28–38.
71. Nakao S, Moriya Y, Furuyama S, Niederman R, Sugiya H. Propionic acid stimulatessuperoxide generation in human neutrophils. Cell Biol Int 1998; 22: 331–337.
72. Nguyen NH, Morland C, Gonzalez SV, Rise F, Storm-Mathisen J, Gundersen V et al.Propionate increases neuronal histone acetylation, but is metabolized oxidatively by glia.Relevance for propionic acidemia. J Neurochem 2007; 101: 806–814.
73. Conn AR, Fell DI, Steele RD. Characterization of alpha-keto acid transport across blood-brain barrier in rats. Am J Physiol 1983; 245: E253–E260.
74. Hara H, Haga S, Aoyama Y, Kiriyama S. Short-chain fatty acids suppress cholesterolsynthesis in rat liver and intestine. J Nutr 1999; 129: 942–948.
75. Le Poul E, Loison C, Struyf S. Functional characterization of human receptors for shortchain fatty acids and their role in polymorphonuclear cell activation. J Biol Chem 2003; 278:25481–25489.
76. Rorig B, Klausa G, Sutor B. Intracellular acidification reduced gap junctioncoupling between immature rat neocortical pyramidal neurones. J Physiol 1996; 490(Pt 1): 31–49.
77. Brass EP. Interaction of carnitine and propionate with pyruvate oxidation by hepatocytesfrom clofibrate-treated rats: importance of coenzyme A availability. J Nutr 1992; 122:234–240.
78. Chandler RJ, Zerfas PM, Shanske S, Sloan J, Hoffmann V, DiMauro S et al.Mitochondrial dysfunction in mut methylmalonic acidemia. The FASEB Journal 2009; 23:1252–1261.
79. Pettenuzzo LF, Ferreira Gda C, Schmidt AL, Dutra-Filho CS, Wyse AT, Wajner M.Differential inhibitory effects of methylmalonic acid on respiratory chain complex activities inrat tissues. International J Dev Neurosci 2006; 24: 45–52.
80. Brusque AM, Borba Rosa R, Schuck PF, Dalcin KB, Ribeiro CA, Silva CG et al. Inhibition ofthe mitochondrial respiratory chain complex activities in rat cerebral cortex bymethylmalonic acid. Neurochem Int 2002; 40: 593–601.
81. Miecz D, Januszewicz E, Czeredys M, Hinton BT, Berezowski V, Cecchelli R et al.Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood-brainbarrier. J Neurochem 2008; 104: 113–123.
82. Celestino-Soper PB, Violante S, Crawford EL, Luo R, Lionel AC, Delaby E et al. A commonX-linked inborn error of carnitine biosynthesis may be a risk factor for nondysmorphicautism. Proc Nat Acad Sci USA 2012; 109: 7974–7981.
83. Scafidi S, Racz J, Hazelton J, McKenna MC, Fiskum G. Neuroprotection by acetyl-L-carnitine after traumatic injury to the immature rat brain. Dev Neurosci 2010; 32: 480–487.
85. Fortin G, Yurchenko K, Collette C, Rubio M, Villani AC, Bitton A et al. L-carnitine, adiet component and organic cation transporter OCTN ligand, displays immuno-suppressive properties and abrogates intestinal inflammation. Clin Exp Immunol 2009;156: 161–171.
86. Geier DA, Kern JK, Davis G, King PG, Adams JB, Young JL et al. A prospective double-blind, randomized clinical trial of levocarnitine to treat autism spectrum disorders. Med SciMonit 2011; 17: PI15–PI23.
87. Rossignol DA. Novel and emerging treatments for autism spectrum disorders: a systematicreview. Ann Clin Psychiatr 2009; 21: 213–236.
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