Laboratoire Philippe Auguste1 119Avenue Philippe Auguste 75011 Paris France Tel: (33)1.43.67.57.00...

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Laboratoire Philipp e Auguste 1 Laboratoire Philippe Auguste 119 Avenue Philippe Auguste 75011 Paris France http:// www . labbio .net Tel: (33)1.43.67.57.00 Fax: (33)1.43.79.00.27 Email : [email protected] Improvement in biological indicators in Autism: properly define clinical improvement Robert Nataf M.D.

Transcript of Laboratoire Philippe Auguste1 119Avenue Philippe Auguste 75011 Paris France Tel: (33)1.43.67.57.00...

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Laboratoire Philippe Auguste 1

Laboratoire Philippe Auguste119 Avenue Philippe Auguste75011 Paris Francehttp://www.labbio.netTel: (33)1.43.67.57.00 Fax: (33)1.43.79.00.27Email : [email protected]

Improvement in biological indicators in Autism: properly define clinical improvement

Robert Nataf M.D.

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Laboratoire Philippe Auguste 2

Nervous system

Malnutrition Neuro-inflammation

Oxidative Stress

Genomes

Environment AutismToxins

Immune system

Detoxification system

GI system

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Biochemical Abnormalities in Autism

• Elevated toxic metals and Xenobiotics• Immune Disorder• Severe oxidative stress & damage

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Urinary Porphyrins: A Biomarker of Environmental Toxicity

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What are the porphyrins?

Porphyrins are cruciform compounds (4 Pyrroles) synthesized by all the cells of the living organism which constitute the active sites of the hemoproteins which transport oxygen Hb & Mb, ensure the energy production Cytochromes A3, B, C and the detoxication of xenobiotic, Cytochromes P450.

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8 enzymes ensures in mitochondrion and cytoplasm, the synthesis of porphyrins: It may be divided into 3 steps :

1. reaction of 2 simple molecules, glycine (from the general amino

acid pool) and succinyl-CoA (from the tricarboxylic acid cycle) to Porphobilinogen.

2. Decarboxylation reaction from Uroporphyrin (8-carboxy) to Coproporphyrin (4-carboxy).

3. The synthesis of heme from 2-carboxyporphyrin through two last biosynthetic pathway enzymes.

PORPHYRINS SYNTHESIS

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-aminolevulinicacid (ALA) synthetase2 = ALA dehydratase3 = uroporphyrinogen I synthetase PBGD4 = uroporphyrinogen III cosynthetase5 = UROD (Uroporphyrinogen decarboxylase)6 = COPOX (Coproporphyrinogen oxidase)7 = Protoporphyrinogen oxidase8 = Ferrochelatase

Heme biosynthetic pathway

M.W. 830 M.W. 786M.W. 742

M.W. 698M.W. 566

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Polychorinated Biphenyl (PCB) UroPArsenic (As) 7cxPAluminium (Al)

Mercury (Hg) 5 cxP PcP

CoP

Lead (Pb) CoP

URO-D = Uroporphyrin Decarboxylase COP-O Coproporphyrin oxidase

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ALA

SUCC

8-CP 5-CP 4-CP 2-CP

Heme

XenobioticsXenobiotics

CPOX

Xenobiotics target in Heme biosynthetic pathway at 3 sides

glycine

ALA-S

+ +

7-CP 6-CP

- --

-

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Xenobiotic compounds

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Aluminium (Al)

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Arsenic (As)

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3 ENZYMES OF THE HEME BIOSYNTHETIC PATHWAY

LEAD TARGETS

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Succinyl-CoA

Kreb Cycle

Pb Glycine

5-Amino-levulinate (ALA)

23137

HEME

Protoporphyrinogen

Coproporphyrinogen

UroporphyrinogenPorphobilinogen

42124

4318

42175

41137

1333

1334 49911

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PORPHYRIN BIOSYNTHETIC PATHWAY PROVIDES 3 MARKERS OF LEAD TOXICITY

ALA DEHYDRATASE  2nd==> ALA (5 Amino Levulinic Acid) in urine and plasma

COP-OXIDASE     6th enz ==> COPROPORPHYRINE in urine (lack of specifcity)

HEME SYNTHASE  8th enz==>  PROTOPORPHYRIN & ratio PP/HEME in blood erythrocytes

Targeted Enzymes Biological Markers

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Lead (Pb)

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UROPORPHYRIN DECARBOXYLASE (MW 80000)

8-carboxy-P or Uro-P

7-carboxy-P 6-carboxy-P 5-carboxy-P 4-carboxy-P or Copro-P

UROPORPHYRIN DECARBOXYLASE POSSESSES 4 DISTINCT SITES OF DECARBOXYLATION

-CO2-CO2-CO2

-CO2

1st site 2nd site 3rd site 4th site

Hg inhibits only the 4th site of decarboxylation generating accumulation 5CXP

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8-CP 7-CP 6-CP 5-CP 4-CP 2-CP Heme

KICP

HgHg

CPOXUROD

CPOX4

Mercury targets CPOX and UROD in Heme biosynthetic pathway

James S. Woods et al.. Texicology Letter 2005….

Promotion in presence of Hg

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Mercury (Hg)

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Urinary Hg = 0.048 µg/l = 0.065 µg/gCr

Age = 3

WO JA

Moderate Hg toxic effect

PCP/URO = 1

5CXP >7CXP

COP increased

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Urinary Hg = 0.41 µg/l = 0.235 µg/gCr

PCP/URO = 1

5CXP > 7CXP

association? Uncoupled COP

Moderate Hg toxic effect

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NI AN

Urinary Hg = 0.11 µg/gCr

Age = 5

PCP = 1. 3 URO

5CXP > 7CXP = 1.5

increased COP

Noticeable Hg toxic effect

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Urinary Hg = 0.337 µg/l = 0.272 µg/gCr

WE RI

Age = 4

High and exclusiveHg toxic effect

High PCP/URO > 4 PCP/5CXP > 5 5CXP/7CXP > 2, repression URO level and Por Synth Rate ?

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SO KE

Urinary Hg = 0.058 µg/gCrAge = 3

PCP twice higher URO

5CXP > 7CXP

high COP

xenobiotics ?

lead ?

High Hg toxic effect

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Urinary Hg = 1.06 µg/gCr

Age = 2

SA SA

probably no up regulated porphyrin synthesis but polyhalogenated uro-D inhibition, and increased Hg specific porphyrins

Estonian child with MENTAL

RETARDATION

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ZH SA

Urinary Hg = 0.312 µg/gCrAge = 2 High Hg toxic effect

Xenobiotics

Up-regulation porphyrin synthesis

PCP twice higher URO

5CXP > 7CXP

high COP

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brothers

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Twin Brothers

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Parents

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High Hg Toxicity

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17/12/0704/07/07

precoproprecopro

Pre-Chelation Post-Chelation

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02/01/0814/11/07

Pre-Chelation Post-Chelation

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25/04/07 29/11/07

Pre-Chelation Post-Chelation

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08/06/07 12/11/07

Pre-Chelation Post-Chelation

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20/06/07 12/10/07

Pre-Chelation Post-Chelation

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20/06/07 12/10/07

Pre-Chelation Post-Chelation

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20/06/07 12/10/07

Pre-Chelation Post-Chelation

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10/07 12/07Pre-Chelation Post-Chelation

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26/07/07 02/11/07

Pre-Chelation Post-Chelation

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15/06/07 12/11/07

Pre-ChelationPost-Chelation

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01/06/07 13/11/07

Pre-Chelation Post-Chelation

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22/08/07 18/12/07

Pre-Chelation Post-Chelation

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Pre-Chelation Post-Chelation

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Nataf R, Skorupka C, Amet L, Lam A, Springbett, Lathe R,

Porphyrinuria in childhood autistic disorder, Implication for environmental Toxicity

Toxicology and Applied Pharmacology (June, 2006)

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Condition/diagnosis M F TotalMean age (yr)

M/F % total% ASD group

Allergy 5 3 8 7,3 1,67 3Asperger 10 1 11 10 10 4,1 5,8

Attention deficit 2 7 9 9,4 0,29 3,3Autism (autistic disorder) 79 27 106 6,4 2,9 39 55,5

Autism+epilepsy 7 2 9 9,3 3,5 3,3Cerebral palsy 6 6 12 8,3 1 4,4

Epilepsy 2 0 2 10 na 0,7Hyperactivity 27 2 29 9,1 13,5 10,7MR+ epilepsy 1 1 2 6 1 0,7

PDD-NOS 51 12 63 6,6 4,3 23,4 33Psychomotor retardation 1 3 4 7,3 0,33 1,5

Rett 0 2 2 2,5 0 0,7Control group 7 5 12 10,3 1,4 4,4 1

TOTAL 198 71 269 7,4 2,8

ASD= 71% of

total sample (M/F=3,34)

269 Study Subjects (2002-2004)

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CTRL MEAN

CTRL 2 x SD

Coproporphyrin levels in urines of children with neurodevelopmental and related disorders

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Laboratoire Philippe Auguste 47

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Elevated urinary Coproporphyrin (COPRO) levels in ASD expressed as absolute values normalised to creatinine (left) or

as an internal ratio with uroporphyrin (URO) (right)

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Precoproporphyrin and pentacarboxy porphyrin: Markers of heavy metal toxicity

0

2

4

6

8

0 2

0

2

4

6

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0 2

Control ASP

(0.62)

(0.56)

Precoproporphyrin plotted against baseline uroporphyrin : the ratio is independent of age-related creatinine variation

0

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(1.09)

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uroporphyrin

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Laboratoire Philippe Auguste 49

0

1

2

3

4

5

ASP PDD-NOS AUT AUT+EPI CTL

Po

rph

yrin

rat

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to

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up

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UROP

7CXP

6CXP

5CXP

COP

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Spectrum of mean porphyrin excess, expressed as a ratio of control group (CTL) value for the different porphyrin subtypes

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Neopterin Marker of Neuroinflammation

Neuroinflammation in Autism

x ybiopterin

OxidativeStress

+ -

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Neuroinflammation appears as a prominent histological feature in brain regions affected by autism.

Mediated by virus vaccine or infectious agents or other factors, neuroinflammation is largely cytokine dependent.

Neopterin, which is a central plate form and a common final pathway in cytokine production and activity, is regarded as

an index of inflammation associated immune activation.

Synthesized through the same teleonomic pathway, reduced Biopterin (BH4), alleviates oxidative damage issued from

neopterin induced immune activation.

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Neopterin (Autist)

Neopterin(control)

Biopterine (Autist)

Biopterine (control)

URINARY NEOPTERIN & BIOPTERIN

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Upon stimulation with the Th1 cell-derived cytokine interferon-g, human macrophages produce neopterin, a 2-amino-4-hydroxy-6-(D-erythro- 1´,2´, 3´-tryhydroxypropyl)-pteridine. Besides,

macrophages also release reaktive oxygen species (ROS) and tumor necrosis factor-a (THF-a).

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Neopterin

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Biopterin is Neopterin’s antidote. 

If Neopterin is constantly increased in autism, Biopterin is most frequently diminished. 

The enlarged differential between these two pterins could be considered as an index of deleterious consequences of

inflammation.

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Autism Control

515

220

500

nmole/mmole Cr

Urinary Neopterine in Autism in 110 Children (Age between 2-11 years old)

100

200

300

400

600

n=159

n=32

Neo

pter

ine

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20/06/07 12/10/07

Pre-Chelation Post-Chelation

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10/07 12/07Pre-Chelation Post-Chelation

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Pre-Chelation Post-Chelation

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Oxidative Stress in Autism

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Underlying reasons :: • Neuroinflammation, • Infectious processes,• Mercury oxidative injury • Gene-Environment interaction issued metabolic imbalance.

If so, assessment should turn to brain damaging oxidative markers recently highlighted in neurodegenerative diseases.

In preliminar results in our laboratory, children  suffering from ASD, exhibit elevated urinary 8-oxo-guanosine(8OHG), RNA oxidation by-product, twice higher than in sibling controls.

Is oxidative stress increased in Autism ?

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8-oxo-deoxyguanosine(8OHdG)

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°OH °O

8-oxo-guanosine(8-OG)

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8-oxo-guanosine(8-OHG)

8-oxo-deoxyguanosine(8OHdG)

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Main pathologies related to 8-oxo-DeoxyGuanosine increase

8-oxo-deoxyguanosineDNA oxidative damage marker

to monitor oxidative stress

8-oxo-deoxyguanosineDNA oxidative damage marker

to monitor oxidative stress

agingaging CancerCancer

IntoxicationIntoxicationCardio-vascular diseasesCardio-vascular diseases

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Main pathologies related to 8-oxo-Guanosine increased

brain degenrative diseases

brain degenrative diseases

Alzheimer DiseaseAlzheimer Disease

Parkinson DiseaseParkinson DiseaseSenile DementiaSenile Dementia

Lateral Amyotrophic Sclerosis

Lateral Amyotrophic Sclerosis

8-oxo-guanosineRNA oxidative damage marker

to asses oxidative stress

8-oxo-guanosineRNA oxidative damage marker

to asses oxidative stress

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RNA oxidation is an early and prominent feature of main brain neurodegenerative diseases

ADPD

Senile Dementia with lewy bodiesALS

MSA-PD 

In AD cytoplasmic RNA oxidation by free radicals released by mitochondria is an early event of perikaryon degeneration

precessing specific neurofibrillar tangles .

RNA oxidation is an early and prominent feature of main brain neurodegenerative diseases

ADPD

Senile Dementia with lewy bodiesALS

MSA-PD 

In AD cytoplasmic RNA oxidation by free radicals released by mitochondria is an early event of perikaryon degeneration

precessing specific neurofibrillar tangles .

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Mitochondria failureMitochondria failure

MtDNA deletionMtDNA deletion

H2O2released in cytoplasmH2O2released in cytoplasm

cytoplasmic iron -> highly reactive oxygen speciescytoplasmic iron -> highly reactive oxygen species

All RNA species (r,t,m) oxidationAll RNA species (r,t,m) oxidation

Protein synthesis impairmentProtein synthesis impairment

Neurodegenrative specific stigmatsNeurodegenrative specific stigmats

Is cytoplasmic RNA oxidation a mitochondria’s failure consequence ?

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Autism

Autism

Control

Control

87

43

35

50

100

8OHG 8OHdG

nmole/gCr

DNA and RNA Oxidative Markers preliminary results in 110 Children (Age between 2-11 years old)

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20/06/07 12/10/07

Pre-Chelation Post-Chelation

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Environment Intoxication?

Inflammation Stress Oxydant

Genetic

Structure Modification

Function Loss

Pathogenic Interactions in Autism??

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Using Urinary Porphyrin, Neopterin

and 8OHdG/8OHG Profile In Chelation Therapy

Using Urinary Porphyrin, Neopterin

and 8OHdG/8OHG Profile In Chelation Therapy

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Urinary Porphyrin profile attests the effectiveness of Chelation Therapy, displaying the reduction of Mecury Responsive Metabolites 5-carboxyporphyrins, Precoproporphyrins, Coproporphyrins and often total Porphyrinuria.

It helps to determine when a patient is cleaned up from their body burden of toxicities.

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5-Carboxyporphyrine

0

5

10

15

20

25

30

35

40

45

0 1 2 3

nmol

e/gC

r

Pre-chelation Post-chelation

n=190age = 1-12 years old

5-carboxyporphyrin

0

2

4

6

8

10

12

14

nm

ol/g

Cr

Pre-chelation

Post-chelation

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Precoproporphyrin

0

5

10

15

20

25

30

35

40

45

nm

ol/g

Cr

Precoproporphyrin

0

10

20

30

40

50

60

70

80

0 1 2 3

nmol

e/gc

r

Pre-chelation Post-chelation Pre-chelation Post-chelation

n=190age = 1-12 years old

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Coproporphyrin

0

100

200

300

400

500

600

Coproporphyrin

0

100

200

300

400

500

600

700

800

900

1000

nmol

e/gC

r

Pre-chelation Post-chelationPre-chelation Post-chelation

n=190age = 1-12 years old

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neopterines

0

200

400

600

800

1000

1200

1400

nm

ol/

gC

r

n=39age = 3-10 years old

pre-chelation post-chelation

neopterins

0

100

200

300

400

500

600

700

nm

ol/g

Cr

post-chelationpre-chelation

As the same time decrease inflammation, as shown by reduction in macrophage activation issued Neopterins.

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Oxidative Stress

0

20

40

60

80

100

120

140

160

nm

ole

/gC

r

8OHdG 8OHG

pre-chelation pre-chelationpost-chelation post-chelation

Oxidative Stress

0

10

20

30

40

50

60

70

80

90

nm

ol/g

Cr 8OHdG

8OHG

Pre-chelation Post-chelation Pre-chelation Post-chelation

n=23age 2 -12 years old

And decreaseoxidative stress , inside nucleus and through cytoplasm, as shown by 8-oxo-Deoxyguanosine (8OHdG) and 8-oxo-Guanosine(8OHG) reduction.

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