Laurette Janak

Autism One May 2009 Part 1 of 2

Laurette.janak@verizon.net
Overview
Part One: the “What”

What has been found?

What it means?
What to do about it?

Part Two: the “How”

How the biochemical

pathways for folate and
glutathione function.
 Metabolic Findings in Parents of
Children with Autism
• 86 autism parents differ from 200
controls in the following:

– Higher homocysteine (Hcy)

– Higher SAH (S-adenosylhomocysteine)
– Lower GSH (glutathione)
– Increased GSSG (oxidized glutathione)

J Autism Dev Disord. 2008 Nov;38(10):1966-75

 Mothers of Autistic Children
J Autism Dev Disord. 2008 Nov;38(10):1966-75

200 Control
46 Case Moms
Moms
Mean
9.8 * 7.4
Homocysteine

Mean SAH 33 * 23

Mean total GSH 5.1 * 7.3

Mean GSSG .32 * .24

* p < 0.001 Case Versus Control Mothers

Risk vs. Causation
Elevated Homocysteine
• What does elevated
homocysteine mean?

• It is a marker for a folic

acid and/or vitamin B12
deficiency. (B-6)

• What are the health

implications of elevated
homocysteine?
 Homocysteine, folate, methylation and
monoamine metabolism in depression
–J Neurol Neurosurg Pyschiatry 2000;69(2):228-32

52% of the depressed persons in this study

had elevated homocysteine
Full text available at:
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1737050&blobtype=pdf
 Depression

“Relatives of autistic individuals have high rates of

major depression and social phobia that are not
associated with the broad autism phenotype and
cannot be explained by the increased stress
associated with raising an autistic child.”
Am J Psychiatry 1999 Apr;156(4):557-63

Full text available at: http://ajp.psychiatryonline.org/cgi/reprint/156/4/557

 Depression
• “Folic acid is a simple method of greatly
improving the antidepressant action of
fluoxetine…. Folic acid should be given in
doses sufficient to decrease plasma
homocysteine.”

– Enhancement of the antidepressant action of fluoxetine by

folic acid: a randomized, placebo controlled trial
• J Affect Disord 2000 Nov;60(2):121-30

• What if the doctor does NOT screen you for

homocysteine before putting you on a SSRI?
Depression Linked to Heart Disease

Depression has been shown

to be an independent
risk factor for coronary artery
disease and vice versa.

–Arch Gen Psyschiatry 2004 Apr;61(4):369-76.

–Eur Heart J. 2004 Jan;25(1):3-9.
–Prostaglandins Leukot Essent Fatty Acids 2004 Apr;70(4):56,349
Cardiovascular Disease
• Elevated homocysteine is an independent risk factor
for vascular disease

– Hyperhomocysteinemia: an independent risk factor for

vascular disease
» N Engl J Med 1991 Apr 25;324(17):1149-55
– Plasma total homocysteine and cardiovascular and
noncardiovascular mortality: the Hordaland Homocysteine
Study.
» Am J Clin Nutr 2001 Jul;74(1):130-6

• Folate and homocysteine metabolism: therapeutic targets

in cardiovascular and neurodegenerative disorders.
» Curr Med Chem 2003 Oct;10(19):1923-9
 Alzheimer’s Disease

• Elevated homocysteine is an
independent risk factor for
Alzheimer's disease.

– Neuroscience 2007 Mar 30;14593):942-54

– N Engl J Med. 2002 Feb 14;346(7):476-83.
– Neuroreport. 2003 Jul 18;14(10):1391-4.
 Parkinson’s Disease
• Disturbances in folate metabolism
have been implicated as a risk factor
for Parkinson’s.
– Int J Mol Med. 2004 Mar;13(3):343-53
– J Neurol Sci. 2003 Mar 15;207(1-2):19-23.
– Neurology. 2003 Feb 25;60(4):690-5.
– Trends Neurosci. 2003 Mar;26(3):137-46.
– Nutr Rev. 2002 Dec;60(12):410-3.
– J Neural Transm. 2002 Dec;109(12):1445-52.
– Int J Geriatr Psychiatry. 2002 Sep;17(9):859-64.
– J Neurochem. 2002 Jan;80(1):101-10.
Michael J. Fox
 Schizophrenia and Homocysteine
• Elevated third trimester homocysteine levels have been
associated with increased risk for schizophrenia in the
offspring.
– Full text available at: http://archpsyc.ama-assn.org/cgi/reprint/64/1/31

• An increased occurrence of schizophrenia has been

reported in parents of children with autism when compared
to control parents.
– Pediatrics. 2008 May;121(5):e1357-62

• In a randomized, double-blind, placebo-controlled,

crossover designed study, 42 schizophrenic patients with
elevated homocysteine were treated with folic acid, B-12,
and B-6
– The treatment resulted in significant clinical improvements
• Biol Psychiatry. 2006 Aug 1;60(3):265-9
 Cancer
“Most families report cancer
in the parents or grandparents,
the most common being
colon cancer.”
–Autism and vaccinations by Mary Megson, MD.
•Wise Traditions Fall 2000

Increased mortality from cancer

in individuals with autism.
–Causes of death in autism.
•J Autism Dev Disord. 2001 Dec;31(6):569-76
Cancer
• Epidemiology studies
have shown an
association of reduced
folate intake with an
increased occurrence
of certain cancers.
 Cancer
Folic acid used as a chemopreventative agent has
shown that results depend on the timing of the folic
acid supplementation. For example:

Folic acid supplementation prior to any

neoplastic lesions was shown to be
protective against colon cancer.

Folic acid supplementation once

neoplastic lesions are present has
been shown to fuel colon cancer
growth.
 Fibromyalgia Myth

• “Fibromyalgia is essentially a benign

disease. It is not a degenerative or
deforming condition, nor does it result in
life-threatening complications.”
– (Patient information: Fibromyalgia from the Cleveland Clinic 6/22/06 Author
Don L Goldenberg, MD published in www.uptodate.com)
FM patients have less total gray matter in their brains
than controls. J Neurosci 2007 Apr 11;27(15):4004-7

–The longer they have had FM, the greater the gray matter loss
–Each year of FM = 9.5 times the gray matter loss as that seen in normal aging

Lower brain grey matter has been found to be associated with:

Lower blood cell folate

Higher homocysteine
Neurosci Lett. 2003 May 8;341(3):173-6.
 Fibromyalgia Myth
(It’s all in your head)
 Fibromyalgia Myth
(It’s all in your head)
• “the results do confirm that there are real metabolic
changes in painful muscles in PF patients.”
– (PF = primary fibromyalgia)
– This study was done on muscle biopsies
• Arthritis and Rheumatism, Vol 29, No. 7 (July 1986)

• Use of P-31 Magnetic Resonance Spectroscopy to Detect

Metabolic Abnormalities in Muscles of Patients with
Fibromyalgia.
– This study reiterates the findings of the 1986 study using MRS
technology instead of muscle biopsy.
– Findings: “impaired mitochondrial function”
• Arthritis & Rheumatism vol 41, No3, March 1998 pp 406-413
 Mitochondria produce energy (ATP)

• Muscle studies in FM have shown

mitochondrial damage.

– Joint Bone Spine. 2006 May;73(3):239-42.

– Ann Rheum Dis. 2004 Mar;63(3):245-51.

– Curr Rheumatol Rep. 2000 Apr;2(2):131-40.

– Z Rheumatol. 1998;57 Suppl 2:47-51.

Cristae - the site of the electron transport chain

Matrix- the site of the citric acid cycle
 Myth
(It’s all in your head)
Using either muscle biopsy or 31P magnetic resonance spectroscopy the
following are found with respect to FM or CFS:

These values are increased: These values are decreased:

Inorganic phosphate (Pi) Phosphocreatine (PCr)
Phosphodiesters (PDE) ATP
Pyruvate Phosphorylation potential (PP)
Lactate Oxidative capacity (Vmax)
Glycolysis Lactate dehydrogenase (LDH)
iNOS Reduced nutritive blood flow during
aerobic exercise
Increased sensitivity to NO induced Number of mitochondria. Altered
suppression of oxidative phosphorylation. morphology of mitochondria
DNA fragmentation Intramuscular collagen
Lipid accumulation Hydroxyproline (in muscle)
Lipofuscin Lower total concentration of the major
amino acids of collagen than in controls (in
muscle)
Oxidative damage to lipids in muscle Acetyl l-carnitine
Activity of the antioxidant enzymes
catalase, glutathione peroxidase and
transferase
Data I collected from multiple studies cited in Pubmed
 Homocysteine and Mitochondria
• Correlation of plasma homocysteine and
mitochondrial DNA content in peripheral blood in
healthy women Atherosclerosis. 2001 Oct;158(2):399-405.

– A physiologic range of total plasma homocysteine (tHcy)

was shown to have the following significant relationship:.

tHcy mtDNA content

 Chronic Fatigue Syndrome (CFS)
and Homocysteine
Is there a connection?
• Elevated homocysteine was found in cerebrospinal fluid of
women who fulfilled the criteria for both FM and CFS.
– There was a significant correlation between the CSF-Hcy levels and
fatigability.
– Scand J Rheumatol 1997;26(4):301-7

• Plasma homocysteine vs cerebrospinal fluid homocysteine

 Parental Oxidative Stress
• Parents of children with autism were
found to have:
– Significantly decreased levels of GSH
(glutathione)
– Significantly increased levels of GSSG
(oxidized glutathione)
– Significantly decreased ratio of GSH/GSSG
• J Autism Dev Disord. 2008 Nov;38(10):1966-75

• This means the parents are under

oxidative stress.
 Oxidative Stress and Psychiatric
Disorders
• A meta-analysis indicates an association of
oxidative stress in the majority of DSM-IV
psychiatric disorders including: autism, Rett’s,
ADHD, schizophrenia, anxiety, and mood
disorders. Full text available at:
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2330073&blobtype=pdf

• “…all these psychiatric disorders might benefit

from a change to a whole-food plant-based diet.”
 Oxidat ive Stress in Chr on ic F at igue
Synd rome
Erythrocyte oxidative damage in chronic fatigue syndrome. 
Arch Med Res. 2007 Jan;38(1):94­8.  
 
Chronic fatigue syndrome is accompanied by an IgM­related immune response 
directed against neopitopes formed by oxidative or nitrosative damage to lip ids 
and proteins. 
Neuro Endocrinol Lett. 2006 Oct;27(5):615­21.  
 
Oxidative stress levels are raised in chronic fatigue syndrome and are associated 
with clin ical symptoms. 
Free Radic Biol Med. 2005 Sep 1;39(5):584­9.  
 
Chronic fatigue syndrome: assessment of increased oxidative stress and altered 
muscle excitabil ity in response to incremental exercise. 
J Intern Med. 2005 Mar;257(3):299­310.  
 
Elevated levels of protein carbonyls in sera of chronic fatigue syndrome patients. 
Mol Cell Biochem. 2003 Jun;248(1­2):93­5.  
 
Effect of natural and synthetic antioxidants in a mouse model of chronic fatigue 
syndrome. 
J Med Food. 2002 Winter;5(4):211­20.  
 
Relationship between musculoskeletal symptoms and blood markers of oxidative 
stress in patients with chronic fatigue syndrome. 
Neurosci Lett. 2003 Jan 2;335(3):151­4.  
 
Elevated nitric oxide/peroxynitrite mechanism for the common etiology of multiple 
chemical sensitivity, chronic fatigue syndrome, and posttraumatic stress disorder. 
Ann N Y Acad Sci. 2001 Mar;933:323­9 
 
Chronic fatigue syndrome: oxidative stress and dietary modifications. 
Altern Med Rev. 2001 Oct;6(5):450­9  
 
Specific oxidative alterations in vastus lateralis muscle of patients with the 
diagnosis of chronic fatigue syndrome. 
Free Radic Biol Med. 2000 Dec 15;29(12):1252­9.  
 
 
Blood parameters indicative of oxidative stress are associated with symptom 
expression in chronic fatigue syndrome. 
Redox Rep. 2000;5(1):35­41.  
Chronic Fatigue Syndrome

• “CFS patients have a lipid profile and oxidant

biology that is consistent with cardiovascular
risk….Supplementation with specific antioxidant
medications might help to ameliorate symptoms
and any potential cardiovascular complications of
the illness.”
– Free Radical Biology & Medicine 39 (2005) 584-589
 Oxidative Stress in Fibromyalgia

Total antioxidant capacity and the severity of the pain i n patients with 
fibromyalgia. 
Redox Rep. 2006;11(3):131­5.  
 
 
Current concepts in the pathoph ysiology of fibromyalgia: the potential 
role of oxidative stress and nitric oxide. 
Rheumatol Int. 2006 May;26(7):585­97.  
 
 
Antioxidant status, lipid peroxidation and n itric oxide in fibromyalgia: 
etiologic and therapeutic concerns. 
Rheumatol Int. 2006 May;26(7):598­603.  
 
 
Free radicals and antioxidants in primary fibromyalgia: an oxidative 
stress disorder? 
Rheumatol Int. 2005 Apr;25(3):188­90.  
 
 
Are advanced glycation end­product­modified proteins of pathogenetic 
importance in fibromyalgia? 
Rheumatology (Oxford). 2002 Oct;41(10):1163­7.  
• Oxidative stress is implicated in endometriosis.
– J Soc Gynecol Investig. 2006 Sep;13(6):390-8
– Reprod Biomed Online. 2006 Jul;13(1):126-34
– Hum Reprod. 2005 Jul;20(7):2014-20.
– Fertil Steril. 2004 Oct;82 Suppl 3:1019-22.

• High rates of autoimmune and endocrine disorders, fibromyalgia,

chronic fatigue syndrome and atopic diseases among women with
endometriosis: a survey analysis Hum Reprod. 2002 Oct;17(10):2715-24.

• “CONCLUSIONS: Hypothyroidism, fibromyalgia, chronic fatigue

syndrome, autoimmune diseases, allergies and asthma are all
significantly more common in women with endometriosis than in
women in the general USA population.”

– Full text online at:

http://humrep.oxfordjournals.org/cgi/content/full/17/10/2715
• Mitochondrial dysfunction and molecular
pathways of disease Exp Mol Pathol. 2007 Jan 17

– “A wide range of seemingly unrelated disorders, such as

schizophrenia, bipolar disease, dementia, Alzheimer's
disease, epilepsy, migraine headaches, strokes, neuropathic
pain, Parkinson's disease, ataxia, transient ischemic attack,
cardiomyopathy, coronary artery disease, chronic fatigue
syndrome, fibromyalgia, retinitis pigmentosa, diabetes,
hepatitis C, and primary biliary cirrhosis, have underlying
pathophysiological mechanisms in common, namely
reactive oxygen species (ROS) production, the
accumulation of mitochondrial DNA (mtDNA) damage,
resulting in mitochondrial dysfunction. Antioxidant therapies
hold promise for improving mitochondrial performance.”
 Homocysteine and Brain Development
• An animal model of maternal elevated
homocysteine produced offspring with:

– Altered fetal brain development

– Reduced learning abilities
– Increased lipid peroxidation in the brain
• Indicates oxidative stress
– Mitochondria are especially sensitive
– Increased brain DNA fragmentation
– Abnormalities mitigated by using melatonin
• J Pineal Res. 2008 Mar;44(2):181-8.
• J Pineal Res. 2007 Oct;43(3):225-31.
• Am J Pathol. 2007 Feb;170(2):667-79.
 Valproic acid
• Is an anti-seizure medication

• Patients on Valproic acid have been shown to have:

– Elevated homocysteine and decreased folate
• Prog Neuropsychopharmacol Biol Psychiatry. 2008 Apr 1;32(3):844-8
• Epilepsy Res. 2006 Oct;71(2-3):229-32
• Seizure. 2006 Mar;15(2):79-85 Hcy
• Eur J Paediatr Neurol. 2000;4(6):269-77
• Epilepsy Res. 2000 Oct;41(3):253-7
• Brain Dev. 2003 Mar;25(2):113-5. folate
• Acta Neurol. Scand. 69:226-231

– Alterations in the glutathione antioxidant enzymes

• J Basic Clin Physiol Pharmacol 2000;11(1):73-81
• Neuropediatrics 1998 Aug;29(4):195-20
• Br J Clin Pharmacol. 2004 Nov;58(5):542-7.
 Valproic acid
• An animal study of valproic acid showed that within
one hour of receiving a single injection of valproate:
– Significant reductions in hepatic SAM/SAH
– Increase in hepatic GSSG (oxidized glutathione)
– Decrease in plasma vitamin B-6
• Full text available at: http://jn.nutrition.org/cgi/reprint/132/9/2737

• A randomized, double-blind, placebo controlled trial

of melatonin add-on therapy in epileptic children on
valproate monotherapy: effect on glutathione
peroxidase and glutathione reductase enzymes
– I recommend you read the full text avaialble
at:
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1884631&blobtype=pd
 Valproic acid
• Gestational valproic acid exposure is used as
an animal model for autism as well as being
implicated in the development of autism in
human fetuses exposed during critical time
points during gestational development.
– Proc Natl Acad Sci U S A. 2007 Aug 14;104(33):13501-6
– Full text available at:
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1948920&blobtype=pdf
Big Question

• Can autism result from

other environmental
exposures or conditions
which cause a similarity
to the abnormal
biochemistry induced
by gestational valproic
acid?
 Abnormal transmethylation/transsulfuration metabolism and
DNA hypomethylation among parents of children with autism.

Control Case Odds ratio

Stratified group
Mothers N (%) Mothers N (%) (95% CI)

SAH > 30 uMol/l 28 (14) 25 (54.3) 7.3 (3.6, 14.8)

SAM/SAH < 2.5 20 (10) 25 (54.3) 10.7 (5.1, 22.5)

tGSH/GSSG < 20 22 (11) 30 (65.2) 15.2 (7.2, 32.1)

SAM/SAH < 2.5

and 3 (1.5) 19 (41.3) 46.2 (12.8, 166.5)
tGSH/GSSG < 20
Autism Dev Disord. 2008 Nov;38(10):1966-75
What are the repercussions of prenatal
mercury exposure
 Toxicol Appl Pharmacol. 2008 Feb 15;227(1):147-54

This study showed that prenatal

mercury exposure altered the
postnatal development of the brain
glutathione (GSH) antioxidant
system.
 Key points from the study
• The alterations in the brain GSH system
endured when cerebral mercury levels
decreased nearly to basal levels.

• Abnormal development of the GSH system

occurred even when prenatal mercury
exposure was so low that it resulted in
brain mercury levels indistinguishable
from those observed in controls.

Toxicol Appl Pharmacol. 2008 Feb 15;227(1):147-54

Can elevated gestational homocysteine
work in a way similar to prenatal
mercury exposure
Symptoms of magnesium deficiency

• Poor sleep patterns, insomnia, restless sleep with frequent awakenings

• Increased sensitivity to noise
• Cognitive disorder, poor memory, confusion, disorientation
• Increase in migraines
• Increased muscular hyperexcitability, muscle twitching, spasms
• Tingling (may be described as zapping), numbness
• Heart arrthymias, palpitations, mitral valve prolapse, angina
• Anxiety, panic attacks
• Depression
• Irritability
• Asthma
• Difficulty learning
• PMS
 Magnesium deficiency causes:
• Hypersensitivity of the NMDA receptor
• Increased histamine release (allergies)
• Decrease in cell mediated immunity
• Increased inflammatory cytokines
• TNF, IL-1, IL-6, substance P
• Increased tendency for seizures
• Increased insulin resistance
• Increased oxidative stress
Magnesium, glutathione & oxidative stress

GSH/GSSG Oxidative stress

Hypertension 1999;34:76-82
 Parental Magnesium
• Magnesium Research 2006; 19(1): 53-62

– Lower RBC-Mg was found in both mothers and fathers of

children with autism

• There was a statistically significant correlation of the mother’s

Mg to that of her child with autism
– > Consistent with altered GSH/GSSG in autistic children and their
mothers

• Behavioral improvement in the child with autism correlated with

increases in RBC-Mg while on Mg supplements. Off
supplement, behavior regressed as RBC-Mg fell to base-line.

• No difference in serum Mg was found

Testing for Mg deficiency
• Serum Mg levels do NOT reflect tissue levels.
• This is very well documented in the medical literature, yet this is the
method most doctors use to measure magnesium status.
– Am Coll Nutr. 2004 Dec;23(6):732S-7S
– Circulation 1995;92:2190-2197 (full text at: http://www.exatest.com/Research.htm)

• RBC-Mg (Red Blood Cell Magnesium)

– Normal RBC-Mg does not rule out tissue deficiency
• Journal of Chronic Fatigue Syndrome, vol 4(2) 1998
• Nutrition. 1997 Apr;13(4):376-7
Testing for Mg deficiency
• Percentage retention of a Mg load in 24 hr urine test
– This test assumes normal kidney function.
– Increased catecholamines cause increased Mg excretion.
– What can increase catecholamines?
• Stress
• Lack of sleep
• Both of these factors are present in many parents.
 Testing for Mg deficiency
• Sublingual buccal cell Mg
– Accurately reflects ion levels in muscle, cardiac tissue and bone
• J Am Coll Nutr. 2004 Dec;23(6):732S-7S
– More information can be found at: www.exatest.com/physicians.htm
Side Note

Sublingual buccal Mg
7 out of 8 children with
autism were found to have
low levels of sublingual
buccal Mg

Data collected with Dr. Paul Cutler

(DAN doctor)
 glycogen
Anaerobic Glycolysis
Glucose 1-phosphate glucose

Glucose 6-phosphate

Fructose 6-phosphate

Fructose 1,6-bisphosphate

Glycolysis occurs in Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate

the cytosol of the cell.
1,3-Bisphosphoglycerate

3-phosphoglycerate
Places where
magnesium 2-phosphoglycerate
is necessary
Phosphoenolpyruvate

Lactate Pyruvate

The citric acid cycle Oxaloacetate Acetyl-CoA Citrate

occurs in the matrix
Malate Isocitrate
of the mitochondria.
Citric acid cycle
Fumarate 2-Oxoglutarate

Succinate Succinyl-CoA

Journal of Nutritional Medicine (1992) 3, 49-59 available at:

 Recommended Reading

I highly recommend you all read:

– Review and Hypothesis: Might Patients with the Chronic

Fatigue Syndrome have Latent Tetany of Magnesium
Deficiency
•Journal of Chronic Fatigue Syndrome, Vol 4(2) 1998
Mildred Seelig, MD, MPH

•The full text can be found on line at:

www.mgwater.com/clmd.shtml

– You will see many commonalities between what you read in

this CFS paper and what is known about autism.
I wanted a quick fix!

But I knew I didn’t

want to take drugs.

So I took supplements.
I was unable to replete my magnesium.

I tried various forms, dosages and routes:

Pills & powders

Baths & topical

IV magnesium
Magnesium…where are you?
 Consuming delicious greens

www.rawfamily.com Great tasting recipes!

 My magnesium, HVA and HIAA all normalized
with greens, but not with supplements!

This made me question the appropriateness of spending

more money on more supplements to improve my health.
 = EDNP
(Junk
Food )
High Calorie

--------------
Increasing
consumption of
EDNP foods -------

Am J Clin Nutr 2000;72:926-36 Increasing

homocysteine
 Serum concentrations of
vitamins A, E, C, and folate
are inversely related to
consumption of high calorie,
nutrient poor foods.
Am J Clin Nutr 2000;72:926-36

While you cannot have a

genetic epidemic, dietary
deficiencies can unmask a
genetic predisposition to injury
from an environmental
exposure.
 It’s all in the packaging!
Magnesium Spinach
Magnesium packaged with:
Protein, calcium, zinc,
sodium,selenium,vit C, vit B-6,
phosphorus,manganese,
thiamin,riboflavin,vit K, niacin,
pantothenic acid, folate,choline,
betaine, vit A, luetin,fiber,alpha
Magnesium citrate tocopherol,zeaxanthin, gamma
tocopherol and many other
phytochemicals

Folate was first isolated in 1941 from spinach. It is named

after the Latin word folium, meaning leaf.
Plasma vitamin C, cholesterol and homocysteine are
associated with grey matter volume determined by MRI
in non-demented old people. Neurosci Lett. 2003 May 8;341(3):173-6.
Lower brain grey matter was associated with:
Lower blood cell folate
Lower plasma vitamin C EDNP food
Higher homocysteine consumption
Higher cholesterol (Junk)
Food
 A word about phytonutrients
• Food and brain function
– Food for the aging mind (Please, please, please read this!)
• www.ars.usda.gov/is/AR/archive/aug07/aging0807.htm

• Reversing the deleterious effects of aging on neuronal

communication and behavior: beneficial properties of fruit
polyphenolic compounds.
• Am J Clin Nutr 2005 Jan;81(1 Suppl):313S-316S
• full text available at http://www.ajcn.org/cgi/reprint/81/1/313S

• Molecular targets of dietary agents for prevention and

therapy of cancer
• Biochemical Pharmacology 71(2006) 1397-1421
Confusion about what to eat?

Watch 90 min video of Colin Campbell at:

http://www.youtube.com/watch?v=4XzDOGZKI68&feature=related
 Nutrient Density
• A nutrient density standard for vegetables and fruits:
nutrients per calorie and nutrients per unit cost.
J Am Diet Assoc. 2005 Dec;105(12):1881-7.

– “Energy density and nutrient density score were negatively

correlated, confirming the widely accepted notion that energy-dense
foods tend to be nutrient-poor.”

– “… fruits and vegetables had the highest nutrient density score

because they were nutrient-rich in relation to their low energy
content.”
FIGURE 4. Relation between energy density and the naturally nutrient rich score for vegetables
and fruit

Drewnowski, A. Am J Clin Nutr 2005;82:721-732

Copyright ©2005 The American Society for Nutrition Used with permission
 Health Equation
• Eat foods high in nutrients and
low in calories.

• Health = Nutrient/Calories
– Dr. Joel Furhman
– Video: The Greatest Diet on Earth
• www.DrFuhrman.com

• Junk Food defined

– low nutrients with high calories
 Gr eens

• Greens have the highest nutrient per calorie ratio of all fruits and
vegetables.
– www.peertrainer.com/diet/nutrient_density.html

• Greens are a good source of magnesium, folic acid and vitamin K.

• “Vitamin K intake may be a marker of a healthy diet because it is

found mainly in green, leafy vegetables”. Only 27% of people in
the United States consume the average daily goal for vit K.
– Food consumption survey analysis from the Human Nutrition Research
Center, part of the Agricultural Research Service. Agricultural
Research/August 2007
• http://www.ars.usda.gov/is/AR/archive/aug07/vitamin0807.htm
 ORAC Values

• ORAC: Oxygen Radical Absorbance Capacity

• Method for measuring antioxidant capacity of foods

• ORAC database for 277 food items at:

– http://www.ars.usda.gov/Services/docs.htm?docid=15866
 Food description Total-ORAC Calories

Apple juice,unsweetened
408 47 Kcal
without added ascorbic acid

Apple, Red Delicious,raw,

2936 48 Kcal
without skin

Apple, Red Delicious,raw

4275 52 Kcal
with skin

Applesauce,unsweetened,
1965 43 Kcal
without added ascorbic acid

Artichoke (globe or French)

6552 47 Kcal
raw

Artichoke (Ocean Mist)

9416 53 Kcal
boiled

Blueberries, raw 6552 57 Kcal

Data derived from: http://www.ars.usda.gov/Services/docs.htm?docid=15866

 As we age, our bodies produce and
accumulate a family of molecules called
Advanced Glycation End-products.

Increasi ng
AG Es
 Gl ucos Protei n
e
Advanced Glycation End-product (AGE)

Gl ucos Li pi d
e s
Advanced Lipoxidation End-product (ALE)

Glycotoxins
 Facts about AGEs

• AGEs cause proteins or lipids to be structurally

altered in such a way as to make the protein or lipid
dysfunctional.
– Hand analogy with cross-linking

• AGEs are proinflammatory and promote oxidative

stress.

• AGEs are associated with pathological conditions.

• Diabetes, renal failure, artherosclerosis, Alzheimer’s,
rheumatoid arthritis, cancer,cataracts, allergic and autoimmune
diseases
 Food derived AGEs
• AGEs derived from food are absorbed and
contribute to serum AGE levels in both animal
and human studies. Ann N Y Acad Sci. 2005 Jun;1043:452-60

Increased
AGEs in
serum
QuickTimeª and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
 Food derived AGEs
• Once thought to only be problematic in persons with diabetes or
renal failure, dietary AGEs are now being shown to cause
inflammation and contribute to inflammatory and degenerative
processes even in healthy individuals.

– Diet-derived advanced glycation end products are major contributors to the body's AGE pool and
induce inflammation in healthy subjects. Ann N Y Acad Sci. 2005 Jun;1043:461-6.

– Circulating glycotoxins and dietary advanced glycation endproducts: two links to inflammatory
response, oxidative stress, and aging. J Gerontol A Biol Sci Med Sci. 2007 Apr;62(4):427-33.
Advanced glycoxidation end products in
commonly consumed foods. J Am Diet Assoc. 2004
Aug;104(8):1287-91
AGE production in foods is temperature and moisture dependent.

•AGEs in a 90 gram serving of chicken breast

–9,000 kU oven frying
–6,700 kU deep frying
–5,250 kU broiling
–4,300 kU roasting
–1,000 kU boiling

Boiling, poaching, stewing vs. dry heat

So up …. .a be tter
ch oic e!
Foods highest in fat contain the most AGEs,
followed by meats, processed carbohydrates and
then lowest in AGE content are vegetables and fruits.

1 lb = 454 grams
But ter 265 kU /g

Chi cken breast broi led 58 kU/ g

Kel logs Rice Kri spi es

18kU /g
Green beans 0. 18 kU /g
Banana 0. 01
kU /g
(not to scale)
J Am Diet Assoc. 2004 Aug;104(8):1287-91
Advanced glycoxidation end products in
commonly consumed foods. J Am Diet Assoc. 2004
Aug;104(8):1287-91

AGEs in whole human milk AGEs in Enfamil Infant Formula

0.05 kU/ml 4.86 kU/ml
“…since AGEs are known immune cell modulators, the introduction of
infant diets, rich in AGE antigens, may account for the rise in
childhood autoimmune diseases such as Type 1 Diabetes.”
 Side Note
Breast-feeding reduces risk of:
Acute otitis media
Non-specific gastroenteritis
Lower respiratory tract infections
Evid Rep Technol Assess . 2006
Atopic dermatitis Apr;(134):1-161.
Asthma (young children)
Obesity
Type 1 and 2 diabetes
Childhood leukemia
Sudden infant death syndrome
Necrotizing enterocolitis
 Hiding AGEs
• Unexpected places for finding food AGEs

– Soy sauce 8,700 AGE, units /15 ml

• (3 teaspoons)

– Classic Coca-Cola 8,500 AGE, units/cup

– Diet Coke (soda) 9,500 AGE, units/cup

Proc Natl Acad Sci USA 1997 June 10; 94(12): 6474-6479
Regular AGE Diet Low AGE Diet

Same foods different

temperatures used
during preparation.

Basel ine compared to 24

months 40% increase in GSH/GSSG
25% decrease in GSH/GSSG

20% increase in 8-isoprostanes 45% decrease in 8-isoprostanes

Decreased AGER1 expression Increased AGER1 expression

Increased RAGE expression Unaltered RAGE expression
Regular lifespan Extended lifespan
(equal to mice on 40% calorie restriction)

Am J Pathol. 2007 Jun;170(6):1893-902. Full text available at:

www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17525257
 Side note: AGEs in autism
• Proteomic studies identified a single nucleotide polymorphism in
glyoxalase I as autism susceptibility factor. Am J Med Genet A. 2004 Nov
15;131(1):11-7.
– Analyses of autopsied autism brains show AGE accumulation.
• These were not brains from aged persons.
– 38% decrease in Glo1 enzyme activity in autism brains versus controls
• Results in accumulation of brain AGEs

• Alterations of circulating endogenous secretory RAGE and S100A9

levels indicating dysfunction of the AGE-RAGE axis in autism. Neurosci
Lett. 2006 Dec 27;410(3):169-73.
– If an AGE binds to a RAGE receptor it initiates oxidative stress and
inflammation.
– esRAGE receptors can act as decoys to prevent AGEs from binding to the
RAGE receptors.
– Peripheral esRAGE levels were significantly reduced in autism.
 Self-perpetuating cycle
Dietary AGEs
of chronic inflammation
Adapted from a slide by Dr. Jill James
(used with permission)

Acute Inflammation (Resolution)

Heavy Metals
Genetics PGJ(2)
PPAR-RXR dimer
Viral infection (downregulates transcription)
Inflammation GSH/GSSG

TNFα OFF-SWITCH
GSH/GSSG NFkB low
ROS IL-6 iNOS
AP-1 NO
IL-1 high
Free
Nitric oxide
Radicals Transcription Cytokines
factors
GSH/GSSG

Th1
Chronic Inflammation Th2
 Di etary Oxi dati v
e stres s
AGEs

Environmental insults
Infection
Genetic polymorphisms
Dietary deficiencies

Ant ioxidan
ts
Advanced glycation end products and RAGE: a common thread
in aging, diabetes, neurodegeneration, and inflammation.
Glycobiology. 2005 Jul;15(7):16R-28R.

Full text available at: http://glycob.oxfordjournals.org/cgi/content/full/15/7/16R

 Break Time (5 Min)

When we return we will be discussing the

“how” mechanisms of the biochemical
pathways involving folate and glutathione.
 The Folate Cycle

Pyrimidine synthesis
Purine synthesis Uracil
Thymine
Adenine & Guanine
Folate deficiency Folate sufficiency
Methionine
DHF
10-formyl-THF
DHFR

5,10-methylene THF THF

B12 MS
MTHFR homocysteine
Methyl THF
Double Strand Breaks

• DNA deletions

• DNA translocations
 The Folate Cycle
Pyrimidine synthesis
Purine synthesis Uracil
Thymine
Adenine & Guanine
Folate deficiency Folate sufficiency
Methionine
10-formyl-THF DHF
DHFR

5,10-methylene THF THF

B12 MS
MTHFR
Methyl THF
homocysteine
 Methionine synthase
Methionine

5,10-methylene THF THF

B12 MS
MTHFR
Methyl THF
homocysteine
CH3THF

Step 1: CH3 THF CH3 Cobalamin (B12)

(Methylcobalamin)

Step 2: CH3 Cobalamin (B12) CH3 homocysteine

(Methionine)
 methionine
Folate cycle Methylation
Cycle
SAM
THF

SAH
B12 MS

CH3-THF homocysteine
(Methyl THF)

MS : methionine synthase
SAM : S-
adenosylmethionine
SAH : S-
adenosylhomocysteine
 Notes on methylation
 CH3 is called a methyl group

 SAM is called a universal methyl donor

 A family of enzymes called methyl transferases serve

as the working force to transfer methyl groups from
SAM onto other molecules
Methyl transferase taxi analogy
  Used in myelin
basic protein

 Convert serotonin to
melatonin
 Convert
norepinephrine to
epinephrine

 Catecholamine
detoxification via COMT
 Methylation in
the liver is a
detoxification pathway
for many
chemicals.
  Gene expression

 Implications for cancer

 Viral silencing
Decreasing SAM/SAH ratio
Inhibits methyl transferase activity
 methionine
Folate cycle Methylation
Cycle
SAM
THF

SAH
B12 MS

CH3-THF homocysteine
(Methyl THF)
B6

cystathionine
Transsulfuration
B6
pathway
cysteine

glutathione
H 2O

H 2O 2 Hydrogen
Peroxide
 Hydrogen
Peroxide

+ Glutathione
 Glutathione Utilization

cysteine

GST Detoxification of
SOD drugs and chemicals
H2O2 GSH
GPx GR

H2O GSSG

GSH: glutathione
GST: glutathione transferase
GSSG: oxidized glutathione
SOD: superoxide dismutase
GR: glutathione reductase
H2O2: hydrogen peroxide
GPx: glutathione peroxidase
 Notes on Glutathione
 Is an antioxidant made in your body

 Converts hydrogen peroxide to harmless water

 Environmental chemicals, heavy metals, dietary

deficiencies and medical drugs can deplete glutathione

 Glutathione has antiviral properties

 Lower levels found in children with autism compared to

control children

 Protects DNA and mitochondria

 What happens if hydrogen
peroxide production exceeds
glutathione production?
Hydrogen
Peroxide
Hydrogen
glutathione Peroxide
Hydrogen
Peroxide
Hydrogen
Peroxide glutathione
Hydrogen
Peroxide
glutathione
Hydrogen
Peroxide
Art by: Christy Domino
 Metals O2- (super oxide)
Pesticides OH (hydroxyl)
NO (Nitric Oxide)
Chemicals OONO- (peroxynitrite)
Infections H2O2 (hydrogen peroxide)
Poor diet
Damage to:
DNA, proteins, lipids
oxidants antioxidants

No oxidative stress
Free radical production in balance with
antioxidants
Environmental insults Oxi da tive
str ess
Infection
Genetic polymorphisms
Poor diet

Environmental insults can severely tip

the scale in those with increased
vulnerability
Ant ioxidants
 Cancer
• Folic acid impacts
cancer risk in the
following ways:

– DNA methylation (gene

expression)

– DNA synthesis
• Uracil and DNA repair

– Detoxification of chemicals
and control of oxidative
stress
• Methylation of chemicals
• Glutathione (GSH)
– GST helps inactivate
chemical carcinogens into
less toxic or inactive
metabolites
 methionine

THF SAM
B12 MS
SAH H
CH3-THF
(Methyl THF) homocysteine H

High and low values of cystathionine

ASD parents compared to
controls. cysteine
GST
SOD L
H2O2 GSH
GPx GR
H
H2O GSSG
thimerosal Does dose
make the
poison
 GSH
GSH

GSH

GSH
H
GS

thimerosal H
GS

GSH GSH

Glutathione (GSH) protects against

thimerosal induced apoptosis (cell death)
Genes Immun 2002 Aug;3(5):270-8
 GSH

Cell Death
GSH

thimerosal

GSH

SAME DOSE of
thimerosal as
in previous A person with
slide! lower levels of GSH
 GSH

QuickTimeª and a
decompressor
are needed to see this picture.

Low glutathione levels can make people

more sensitive to DNA damage from a
variety of mutagenic environmental
exposures.
A s pecia l Th ank Yo u to
Dr. J ill James
Dr. P aul Cutle r
My fr iend Pa ula
Teri A rr anga
Ray J anak
And to yo u…t he pare nts !