COUPLED DYSBIOSIS & METABOLIC DYSFUNCTIONS IN AUTISM –POSSIBLE REMEDIES

Jon B. Pangborn, Ph.D., F.A.I.C., C.C.N.

TODAY’S ASSIGNMENT:

To explore how impaired methionine-to-taurine metabolism and reduced methylation, together with infection, impact autism. We will consider possible natural and nutritional remedies.

DISCLOSURES

• Father of formerly autistic child – now adult• Left chemical engineering 29 years ago to investigate

biochemistry and autism• Past President, Doctor’s Data Laboratory• Klaire Labs Consultant 1981-1999• Great Smokies Diagnostic Lab (Genova) 1995-2005• Cofounder: Defeat Autism Now! 1995• Coauthor: Autism: Effective Biomedical Treatments• Consultant: Kirkman Labs since 2000• Senior Scientific Consultant, ARI

IN PERSPECTIVE• There are many different autisms:

Primarily Genetic Mostly acquiredInherited faultstoxic/infectious stressors

• Considering ASD people as a group, there are cell-wide and extracellular anomalies.

• Don’t get hung up on one enzyme/protein, one cell compartment, one gene, one epigenetic process, one cytokine, one toxicant or infectious agent or any single theory or model.

TODAY’S FOCUS

• Deficient methylation of lipids adjacent to neuronal receptors can cause loss of neuronal and network synchrony. See: Deth, Molecular Origins of Human Attention Kluwer Acad Pub, 2003.

• Methionine metabolism can be disordered in multiple ways such that methylation is deficient, oxidant stress occurs, and an inflammation becomes a persistent, maladaptive condition.

• Mitochondrial dysfunction is probable when vitamin B12 (as AdoCobalamin) and carnitine are deficient and maybe when Clostridia dysbiosis occurs.

TODAY’S FOCUS2

• Xenobiotic (e.g. organophosphate) and elemental (e.g. mercury) toxicants can be contributory.

• Many infectious agents and foreign substances can lead to oxidant stress when taurine is deficient.

• There are nutritional aids for: methylation, reducing inflammation, mitochondrial support and alleviating dysbiosis.

SAM

SAH

MTase

SAHH

Homocysteine B6 CBS

Methionine Transsulfuration to Cysteine and Glutathione

Cystathionine

Cysteine

GSH GSSG

Methionine

Adenosine

B6

5-CH3THF

THF

B12MS5,10-

CH2THFMTHFR

Cell Methylation1

1

2

3

Folate Cycle

Methionine Cycle

Transsulfuration Pathway SJJ

Methylation Potential(SAM/SAH)

2

3 Antioxidant Redox Potential (GSH/GSSG)

CYSTEINE TO TAURINE (main route)From cystathionine GSH cysteineDietary cystine cysteine sulfinic a GSH GSCys dioxygenase

decarboxylase P5P

Stable OCl-

Chloroamine taurine hypotaurine phagocyte oxygenase

CELL MEMBRANETRANSCOBALAMIN

OHCbl CYTOSOL MeH4folate methionine

GSHLysosome OHCbl GSCbl Cbl MeCbl reductase MS H4folate homocysteine

reductase ATP

PROPIONATE MMA AdoCbl SUCCINATE CARNITINE mutase

C-P COMPLEX C.A. cycle MITOCHONDRION

CARNITINE FORMATION

3 SAM L-lysine trimethyl-L-Lysine 3 methylations CYTOSOL

MITO MEMBRANE MITO

4 biochemical transformations

L-carnitine requiring P5P, NAD, O2, ascorbate and α-ketoglutarate x 2

CARNITINE

CH3

CH3―+N― CH2―CH―CH2―COOH

CH3 OH

ACTIONS OF CARNITINE

• Using a translocase transporter, CARNITINE escorts fatty acids (as fatty-acyl carnitine) through the inner mitochondrial membrane.

• Combines with excesses of organic acids (propionic, isovaleric, 3-methylglutaric, valproic, others) to accomplish a detoxication function.

REVIEW: FOUR INTERRELATED PARTS OF BIOCHEMISTRY

1. Methionine → SAM, methylation →→ cysteine with some homocysteine recycle using methyls provided by folate to Cbl on MS.

2. Cysteine →→ taurine or cysteine →→ GSH Taurine scavenges OCl- GSH neutralizes H2O2 and

during cellular immune and detoxifies and makes B12

defense activities. work.

3. B12 →→GSCbl: cytosolic homocysteine → methionine and mitochondrial cleanup of propionate via MMA

4. Carnitine, needs methylation for formation, also cleans up propionate and other excess organic acids.

WHAT’S GOING WRONG?SO MUCH THAT WE HAVE TO STUDY IT PIECEWISE

METHIONINE → SAM → S-ADENOSYLHOMOCYSTEINE methylation

Reduced methylation causes varying deficiencies of –melatonin choline/TMG/DMGcreatine methylated catecholaminescarnitine methylated CG pairs on DNA

methylated fatty acids

Remedies: supplements of melatonin, creatine, L-carnitine, TMG, DMG. See ARI Publication 34.

CAN A VIRUS IMPAIR METHYLATION IN NEURONS?

Note: Documentation in rat brain cells of reduced neuronal receptor phospholipid methylation during paramyxovirus infection (measles, mumps) – Münzel and Koschel, Proc. Natl. Acad. Sci. (USA) 79 (1982) 3692-6.

Β-adrenergic receptors affected – loss of catecholamine-stimulated methylation

SAH HOMOCYSTEINE (Hcy) METHIONINE ADENOSINE

CYSTATHIONINE CYSTEINE

1. Adenosine accumulates, SAH accumulates, SAH/SAM ratio increases, methylation slows. Adenosine elevated in ASD, 20% per plasma, 33% per RBCs.

2. Adenosine Deaminase (ADA) Binding Protein = DPP4 or CD26 is inhibited by: mercury, antimony, lead, orga-nophosphates, streptokinase, casomorphin peptides.

See Püschel et al. Eur J Biochem 126 (1982) 359-65 Vojdani et al., Int.J.Immunopath Pharmacol 16 3 (2003)

189-99

SAH HOMOCYSTEINE (Hcy) METHIONINE ADENOSINE

CYSTATHIONINE CYSTEINE

3. Genetic variants for ADA, methionine synthase, formation of cystathionine and methylated folate = risk factors for ASD.

4. Hcy → Methionine hindered by oxidation of reduced cobalamin (Co+1)

5. Some Hcy tied up as Hcy thiolactone if PON (paraoxonase, Hcy thiolactonase) weak – organophos-phate toxicity; 40-fold activity variance in humans

6. Functional P5P coenzyme inadequacy

SAH HOMOCYSTEINE (Hcy) METHIONINE ADENOSINE

CYSTATHIONINE CYSTEINE

REMEDIES FOR POSSIBLE PROBLEMS• Methylcobalamin and folinic acid• B6/P5P, Zinc, Magnesium, Melatonin• Trimethylglycine, Dimethylglycine• Diet and detoxification (helps ADA Binding Protein –

DPP4)• Antioxidant nutrients, digestive enzymes• Not recommended: Methionine, SAM, Cysteine, NAC

CYSTEINE TAURINE

1. Cysteine dioxygenase downregulated in autism (inflammation); see studies by Waring.

2. Functional P5P coenzyme inadequacy

3. Urinary loss of taurine due to beta-aminoaciduria, mostly elevated beta-alanine –

Bacterial dysbiosis Anserine and carnosine Pyrimidine disorder, PDD, see studies by Page

CYSTEINE TAURINE

REMEDIES FOR POSSIBLE PROBLEMS• Antiinflammatory meds, digestive aids• B6/P5P, zinc, magnesium, melatonin• Antioxidant nutrients• Taurine, 100-250 mg/day with breakfast

RELEASE OF OXIDANTS BY PHAGOCYTIC NEUTROPHILS (PMN LEUKOCYTES) WHEN TAURINE

IS INADEQUATE

Phagocyte – a cell that ingests microorganisms, foreign particles, other cells, and kills, digests or decomposes them.

Oxidant killing is a normal, body-defense mechanism:Respiratory Burst O2

-, H2O2, OCl-

Taurine limits collateral damage by scavenging OCl-

See Dinauer et al., Chapt 189 in Scriver et al., Eds., The Metabolic and Molecular Bases of Inherited Disease, 8th Ed.

SEE: DISTURBANCES OF AMINO ACID METABOLISM: CLINICAL CHEMISTRY AND DIAGNOSIS, Hans J.

Bremer, et al., Urban & Schwarzenburg, 1981 p225

Table B24. Free amino acids in human polymorphonuclear leukocytes (Houpert et al. 1976)Amino Acid nmoles/million cells

Taurine 15.47Aspartic acid 1.52Alanine 1.51Lysine 1.36Glutamic acid 1.29

Astrocytes: Taurine also the most abundant AA. See Urenjak et al. J.Neurosci 13, 3 (1993) 981-89

WHAT PROVOKES PHAGOCYTIC ACTIVITY AND OXIDATIVE PROCESSES?

• Bacteria (staph, strep, Salmonella, Enterobacter, Proteus, Klebsiella, Microbacteria, others)

• Yeasts (Candida, Aspergillus)• Wheat germ agglutinin, substances that “clump” cells• Fine particles, organic & inorganic, engine & powerplant

exhausts• Fluorides• Some viruses (naked virus w/o lipid envelopes – rhino,

papilloma, rotoviruses)

CLOSTRIDIA OVERGROWTH

• Anaerobic organism; can’t assay by standard culture, often identified by chemical markers: propionic acid, phenyl & hydroxyphenyl propionates, indole propionate, phenol, para-cresol, acetic acid, acetates, phenyllactate.

- See Elsden et al. Arch.Microbiol 107,3 (1976) 283-8

• Many types: C.difficile, propionicum, tetani (credit to Ellen Bolte, 1996), sporogenes, botulin, and many more.

• Propionic acid and propionates induce autistic behavior in rats – See work of Derrick MacFabe.

CLOSTRIDIUM DIFFICILE: ANTIBIOTIC RESISTANT STRAIN

• First public notice: Warrey & Pipin - Lancet 2005; Cloud & Kelly – Curr Opinion Gastroenterol 23,1 (207) 4-9; Baines & O’Connor – J.Microb.Chemother 62,5 (2003) 1078-85; Spigaglia et al. PMID 18480338; Saxon et al. PMID 18710908

• Vancomycin sends it to spore form; later it comes back with a vengeance

• Ciprofloxacin, Levofloxacin and 20 more antibiotics (esp. fluoroquinolones) have no lasting effect. “What almost kills it, makes it stronger.”

NATURAL ANTIBIOTIC FUNCTION IS A TWO-PRONGED ATTACK

A. Toxic killing agents, plusB. Agent that disables organisms’ defenses

See: Proc.Natl.Acad.Sci (USA) 97 (2000) 1433Drs. Lewis (Tufts) and Stermitz (Col. State U)Studied Barberry: berberine + 5’methoxyhydnocarpin

WHAT HERBALS HAVE NATURAL ANTIBIOTIC ACTION AGAINST CLOSTRIDIA?

• Thyme, oregano, cumin (at low levels). See Dorman & Dean, J. Appl.Microbiol. 88 (2000) 308-16

• Curcumin (turmeric) provides antioxidant protection, lowers cytokines (inc. TNF-α), regulates COX-2 and downregulates NF-Kappa B.

• See –Chainani-Wu J.Alt.Complement Med 9 (1) 2003 161-8Bundy, Walker et al. Prac Nutr Soc 63 2004 126AGuo, Kuo et al. Biochem Pharmacol 680 2004 51-61Menon & Sudheer Adv Exp Med Biol 575 2007 105-25

WHAT PROBIOTICS REDUCE OR HELP TO KILL C. DIFFICILE?

Saccharomyces boulardii is effective for C. difficile disease. S. boulardii and Lactobacilus rhamnosis both reduce antibiotic-associated diarrhea.

See: McFarland LV “Meta-analysis of probiotics for the prevention of antibiotic-associated diarrhea and the teatment of Clostridium difficile disease” Am J Gastroenterol. 2006 Apr: 101 (4) 812-22

Segarra-Newnham M “Probiotics for C-difficile-associated diarrhea: focus on L. rhamnosis GG and S. boulardii” Ann.Pharmacother 2007 Jul; 41 (7): 1284-7

WHAT PROBIOTICS REDUCE OR HELP TO KILL C. DIFFICILE?

Lactobacillus plantarum administration to antibiotic-treated patients reduces colonization of C. difficile.

See –Plummer S, Weaver MA et al. “C. difficile pilot study:

effects of probiotic supplementation on the incidence of C. difficile diarrhea” Int Microbiol. 2004 Mar; 7 (1): 59-62

Klarin B, Wulft M et al. “L. plantarum 299v reduces colonization of C. difficile in critically ill patients treated with antibiotics” Acta Anaesthesiol Scand. 2008 Sep; 52 (8): 1096-102

SHORT HISTORY OF MEDICINE

Problem: I have an earache….

2000 B.C. Here, eat this root.1000 A.D. That root is heathen. Here, say this prayer.1850 A.D. That prayer is superstition. Here, drink this

potion.1940 A.D. That potion is snake oil. Here, swallow this

pill.1985 A.D. That pill is ineffective. Here, take this

antibiotic.1997 A.D. That antibiotic ruins your own immunity.

Here, eat this root.