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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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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0
10
20
30
40
50
60
70
80AL
LER
GY
ASPE
RG
ER
ATTE
NTI
ON
DEF
ICIT
AUTI
SM
AUTI
SM +
EPI
LEPS
Y
CER
EBR
AL P
ALSY
EPIL
EPSY
HYP
ERAC
TIVI
TY
MR
+ E
PILE
PSY
PDD
-NO
S
PSYC
HO
MO
TOR
RET
RET
T
CO
NTR
OL
Gou
RP
Co
pro
po
rph
yri
n (
mic
rom
ol/m
ol C
RT
)
n=2
n=2
n=2
n=4
CTRL MEAN
CTRL 2 x SD
Coproporphyrin levels in urines of children with neurodevelopmental and related disorders
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0
5
10
15
20
25
AS
P
AU
T
AU
T+E
PI
CT
RL
CO
PR
O/U
RO
rati
o
0
10
20
30
40A
SP
AU
T
AU
T+E
PI
CT
RL
Co
pro
po
rph
yri
n )
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
8
0 2
Control ASP
(0.62)
(0.56)
Precoproporphyrin plotted against baseline uroporphyrin : the ratio is independent of age-related creatinine variation
0
1
2
3
4
5
6
7
8
0 2 4
AUT +EPI
0
1
2
3
4
5
6
7
8
0 2 4
AUT
(1.09)
(1.28)
uroporphyrin
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0
1
2
3
4
5
ASP PDD-NOS AUT AUT+EPI CTL
Po
rph
yrin
rat
ios
to
con
tro
l gro
up
val
ues
UROP
7CXP
6CXP
5CXP
COP
PRECOP
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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