Clostridia metabolites inhibit human dopamine-beta-hydroxylase,

increasing toxic dopamine metabolites and severe oxidative

stress in autism

William Shaw PhDThe Great Plains Laboratory,Inc

www.gpl4u.com

Dialogues Clin Neurosci. 2011;13:55-62.

Autism Insights 2010:2 1–11

Nutritional Neuroscience 2010 Vol 13 No 3: 1-10William Shaw

Structure of 3-(3-hydroxyphenyl)-3-hydroxypropionic acid

HO

CHOHCH2COOH 123

1

23

4

5 6

Phenyl group

Hydroxyl group

Propionic acid group

Data from previous Shaw article

• “It was observed that mentally ill patients, in general, seem to excrete much larger amounts of HPHPA than do most normal people.”

• Most patients with mental retardation excrete very low amounts of HPHPA.

Journal of Biological Chemistry 225:269-278,1957

Increased Urinary Excretion of Analogs of Krebs Cycle Metabolites and Arabinose in Two Brothers with Autistic Features.

W Shaw. Clin Chem 41:1094-1104, 1995.

HPHPA

HPHPAHPHPA

Child psychosis during hospitalization(simulated from memory)

• C. difficile-pseudomembranous colitis• C. sporogenes• C. botulinum-food poisoning• C. mangenoti• C. ghoni• C. bifermentans• C. caloritolerans

Clostridia species that produce HPHPA precursors

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

1600.00

1800.00

2000.00

N = 14

N = 30

N = 211

CONTROLMALE

INFANTS

CONTROLMALE

CHILDREN AUTISTICMALES

3-(

3-h

yd

rox

yp

he

ny

l)-3

-hy

dro

xy

-p

rop

ion

ic a

cid

(M

MO

L/M

OL

CR

EA

TIN

INE

)

2 - 13 YRS 2 - 13 YRS3-(3

-hyd

roxy

phen

yl)-

3-hy

drox

ypro

pion

ic

acid

HPH

PA -m

mol

/mol

cre

atini

ne

Controlinfants

Control boys2-13 years

Autistic Boys2-13 years

Figure 2. Distribution of values for HPHPA Clostridia metabolite in urine samples of male infants, control

boys, and boys with autism.

Main brain neurotransmittersNorepinephrine and dopamine

Catecholamine Synthesis

Mainly adrenalgland

Dopamine

Norepinephrine

Catabolism of Dopamine

Organic acid test

Norepinephrine CatabolismOrganic acid test

                                                              

Endoscopy of colon of patient with mild Clostridium difficile overgrowth

Endoscopy of colon of patient with severe Clostridium difficile overgrowth-

pseudomembranous colitis

Formation of p-cresol from tyrosine by Clostridia bacteria

tyrosine 4-hydroxy-phenylacetic

p-cresol p-cresol sulfate4-hydroxy-phenylpyruvic

14728–14733 PNAS August 25, 2009 vol. 106 no. 34

PST

CH2CHCOOH

NH2

CH2CHCOOH

NH2

HO

tyrosine

CH2CHCOOH

NH2

HO

microbial

tyrosine analog

CH2CH2COOH

CH2CH2COOH

HO

NH2

phenylpropionic acid

NH2

3-hydroxyphenyl-propionic acid

CH2CHCOOH

NH2

HO

3,4-dihydroxyphenylAlanine (DOPA)

HO

microbial

dopamine

norepinephrine

human beta-oxidation

CHOHCH2COOH

HO3-(3-hydroxyphenyl)-3-hydroxypropionic

acid (HPHPA)

HO

COOH

3-hydroxybenzoic acid

glycine

3-hydroxy-benzoylglycine(3-hydroxyhippuric)

microbial

human beta oxidation enzymes

phenylalanine phenylalanine hydroxylase

tyrosine hydroxylase

CH2COCOOHHO

4-hydroxyphenyl-pyruvic acid

CH3HO

4-cresol

HVAVMA

epinephrine

Critical effect of intestinal bacteria on brain neurotransmitters

Organic acid test

Organic acid test

Organic acid test

Intestine

** = products 0f Clostridia

* *

**

***

**

***

*

****

*******

Blood vessels

*******

**

************

*

*

********

***

*****

** * * ****

Urine cup

Kidney

* B***

*

***

***

* B

***************

*****

****

****

****

****

*******

**********B= bacteria (beneficial)

**** *

* * *

* *

=Clostridiabacteria

Brain

Body

*

Effect of HPHPA on brain neurotransmitters

HPHPAMmol perMolcreatinine

Date

HVAMmol per Molcreatinine

(Dopamine)Clostridia

Effect of HPHPA on brain neurotransmitters

Date

DopaminemetaboliteHVAMmol per Molcreatinine

11/18 2/26 6/6 9/14 12/23 4/10

5

10

15

20

25

30

0

1

2

3

4

5

6

7

VMA (norepi)

NorepinephrineMetaboliteVMAMmol per molcreatinineHVA (dopamine)

Effect of HPHPA on brain neurotransmitters

Date

11/18 1/7 2/26 4/17 6/6 7/26 9/14 11/3 12/23 2/110

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0

1

2

3

4

5

6

7

8

HPHPAMmol perMolcreatinine

HVA/VMAIndicator ofDopamine to Norepinephrineratio

Linan Chen, et al(2008) Unregulated cytosolic dopamine causes neurodegeneration associated with oxidative stress in mice. J. Neurosci. 28, 425–433

• Dopamine is a very reactive molecule compared with other neurotransmitters, and dopamine degradation naturally produces oxidative species.

• More than 90% of dopamine in dopamine neurons is stored in abundant terminal vesicles and is protected from degradation.

• However, a small fraction of dopamine is cytosolic, and it is the major source of dopamine metabolism and presumed toxicity.

• Cytosolic dopamine undergoes degradation to form 3,4-dihydroxyphenylacetic acid (DOPAC) and HVA as well as hydrogen peroxide via the monoamine oxidase pathway.

Figure 1. Toxicity of excess dopamine

Homovanillic acid (HVA)Dihydroxyphenylacetic(DOPAC)

Linan Chen, et al(2008) Unregulated cytosolic dopamine causes neurodegeneration associated with oxidative stress in mice. J. Neurosci. 28, 425–433

• Alternatively, dopamine undergoes oxidation to form superoxide, hydrogen peroxide, and o-quinone or reacts with cysteine residues on glutathione or proteins to form cysteinyl-dopamine and cysteinyl-DOPAC conjugates.

• These biochemical abnormalities caused by excess dopamine may cause severe neurodegeneration of neural pathways that utilize dopamine as a neurotransmitter.

Prevalence of Clostridium difficile in the gastrointestinal tract of hospitalized children under two years of age. Med Dosw Mikrobiol; 2010;62(1):77-84 (Poland)

• 178 fecal samples of children aged 2 months to 2 years,hospitalized in 2003-2006 were examined for the presence of toxin A/B of C. difficile.

• Toxigenicity of strains was confirmed using PCR.• Susceptibility to antimicrobials was determined. • The percentage of children infected with C.

difficile was 68.6%.• All strains were susceptible to Vancomycin and

metronidazole(Flagyl)

Summary • Clostridia metabolites HPHPA, 4-cresol, and 4-

hydroxyphenylacetic are toxic because they inhibit key enzyme that converts dopamine to norepinephrine

• Toxic dopamine metabolites use up glutathione and person is much more susceptible to other toxic chemicals like mercury, pesticides, antibacterial soaps

Acta psychiatr belg 80:249-265,1980

Main dopaminemetabolite HVAmmol/mol creatinine

p<0.01

B. Garreau et al. Disturbances in dopamine metabolism in autistic children: results of clinical tests and urinary dosages of homovanillic acid (HVA). Acta psychiatr belg 80:249-265,1980.

• Dopamine metabolite (homovanillic acid or HVA) in urine was more elevated in children with autism than in PDD and the severity of symptoms was related to the concentration of HVA

• High dopamine production in brain is associated with repetitive, stereotypical, obsessive, compulsive behaviors

• The effects of norepinephrine are alertness and arousal, and influences on the reward system. Norepinephrine is important for the exploratory behavior essential for learning relations between sensory input, decision processing, motor output, and behavioral feedback.

Organic Acids Test: Great Plains Lab

Organic Acids Test: Great Plains Lab

HVA/VMA= 2.58 (Excess dopamine)

Before Treatment

After Treatment

HVA/VMA= 1.38

Factors involved in autism, Clostridia toxicity measured in urine organic acid test

• HVA-(homovanillic acid)-Major metabolite of dopamine, a major brain neurotransmitter associated with abnormal autistic behavior when it is elevated

• VMA-(vanillylmandelic acid)- Major metabolite of norepinephrine- important for the exploratory behavior essential for learning relations between sensory input, decision processing, motor output, and behavioral feedback.

Factors involved in autism, Clostridia toxicity measured in urine organic acid test

• 4-hydroxyphenylacetic- Clostridia metabolite that is a phenol that is detoxified by phenol sulfo-transferase (PST), leading to increased susceptibility to acetaminophen toxicity. 4-Cresol and HPHPA are also PST inhibitors.

• HVA /VMA ratio –indicates whether there is a healthy balance in the brain between norepinephrine and dopamine

Factors involved in autism, Clostridia toxicity measured in urine organic acid test

• Pyroglutamic acid- high values indicate deficiency of glutathione, a major cause of dopamine toxicity and increased susceptibility to most environmental chemicals

• 4-Cresol- Major metabolite of Clostridium difficile- blocks dopamine beta hydroxylase leading to excess dopamine and abnormal behavior

• HPHPA-Major metabolite of multiple Clostridia species- blocks dopamine beta hydroxylase leading to excess dopamine and abnormal behavior

Clinical usefulness of Clostridia treatments

• Schizophrenia• Psychosis• Depression• Chronic fatigue• Tics, Tourette’s• Autism

• ADD, ADHD• Obsessive compulsive

disorder (OCD)• Seizure disorders• Gastrointestinal

disorders, diarrhea, constipation,Crohn’s disease,colitis

Effects of 3-hydroxyphenylalanine in rats

• Headweaving• Predominantly backward walking• Wet dog shakes• Hyperactivity• Hyper-reactivity

Dyck LE, Kazakoff CW, Dourish CT. The role of catecholamines, 5-hydroxytryptamine and m-tyramine in The behavioural effects of m-tyrosine in the rat. European Journal of Pharmacology 1982; 84(3-4): 139-149

Two months of nystatin and Lactobacillus acidophilus GG therapy in a child with autism

Candida krusei stool

Yeast tartaric urine*

Lacto-bacillus stool

Clostridia HPHPA urine*

Before

4+

993

0

3265

After

0

1

4+

174

normal range

0-1+

0-15

3+ - 4+

0-150* mmol/mol creatinine

Effect of anti-Clostridia therapy on urine excretion of HPHPA* in

young woman with acute psychosis-auditory

hallucinations patient normals During acute 7489 0-150 psychosis After treatment 673 0-150 ( depressed but no psychosis) *mmol/mol creatinine

patient normals Before treatment 1444 0-150

After treatment 13 0-150

*mmol/mol creatinine

Effect of anti-Clostridia therapy on urine excretion of HPHPA* in woman with

depression and chronic fatigue

Treatments for Clostridia bacteria• Vancomycin-oral-not intravenous-5-10 mg/Kg/day div into 3

doses• Flagyl (metronidazole)-30 mg/Kg/day div into 3 doses-10 days• Lactobacillus acidophilus GG-10 -100 billion per day • Alternate name Lactobacillus rhamnosus• (Only bacteria probiotic patented for use in control of

Clostridia)-Culturelle-VSL #3• Saccharomyces boulardi (yeast)• Micellized or IV glutathione or n-acetylcysteine to increase

brain glutathione and reduce neurotoxic dopamine metabolites

• High protein diet (phenylalanine, tyrosine) may increase production of toxic Clostridia metabolites

Summary of Clostridia dopamine abnormalities in autism

• Elevated HPHPA and similar phenolic compounds from multiple species of Clostridia are elevated in more than two-thirds of children with autism.

• Elevated HPHPA and cresol inhibit the conversion of dopamine to norepinephrine, resulting in marked increase in dopamine.

• Dopamine metabolites deplete brain glutathione and also produce marked excess of oxygen superoxide free radicals, 2500 molecules for each dopamine metabolite

• Brain and sympathetic nervous systems normally using norepinephrine switch to dopamine, drastically altering brain and sympathetic nervous system function

Summary of Clostridia dopamine abnormalities in autism

• Presence of this abnormality is determined with HPHPA, cresol, and other phenolic compounds in the Great Plains organic acid test

• Stool testing not useful since HPHPA producing and Non-HPHPA species are not differentiated

• Glutathione depletion by excess dopamine determined by pyroglutamic acid marker in organic acid test.

• Clostridia treated for 10-14 days with vancomycin or flagyl,followed by several months high dose probiotics-culturelle or VSL-3

• Repeat testing every 3 months to prevent recurrence