Medical Management of the Improving Autistic Child
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Improvement in biological indicators in Autism: properly define clinical improvement
Robert Nataf M.D.
Laboratoire Philippe Auguste 2 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 : contact@labbio.net
Laboratoire Philippe Auguste 1
Nervous system
Malnutrition Neuro-inflammation
Environment
Toxins Oxidative Stress
Autism
GI system
Genomes
Immune system
Detoxification system
Laboratoire Philippe Auguste 2
Biochemical Abnormalities in Autism
• Elevated toxic metals and Xenobiotics • Immune Disorder • Severe oxidative stress & damage
Laboratoire Philippe Auguste
3
Urinary Porphyrins: A Biomarker of Environmental Toxicity
Laboratoire Philippe Auguste
4
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.
Laboratoire Philippe Auguste
5
PORPHYRINS SYNTHESIS
8 enzymes ensures in mitochondrion and cytoplasm, the synthesis of porphyrins: It may be divided into 3 steps : • reaction of 2 simple molecules, glycine (from the general amino acid pool) and succinyl-CoA (from the tricarboxylic acid cycle) to Porphobilinogen. • Decarboxylation reaction from Uroporphyrin (8-carboxy) to Coproporphyrin (4-carboxy).
6
Laboratoire Philippe Auguste
3. The synthesis of heme from 2-carboxyporphyrin through two
M.W. 830
M.W. 786
M.W. 742
M.W. 566
Heme biosynthetic pathway
Laboratoire Philippe Auguste
1 = δ-aminolevulinicacid (ALA) synthetase 2 = ALA dehydratase 3 = uroporphyrinogen I synthetase PBGD 4 = uroporphyrinogen III cosynthetase 5 = UROD (Uroporphyrinogen decarboxylase) 6 = COPOX (Coproporphyrinogen oxidase) 7 = Protoporphyrinogen oxidase 8 = Ferrochelatase
M.W. 698
7
Polychorinated Biphenyl (PCB) Arsenic (As) Aluminium (Al)
UroP 7cxP
Mercury (Hg)
5 cxP PcP CoP
Lead (Pb)
CoP
Laboratoire Philippe Auguste
URO-D = Uroporphyrin Decarboxylase
COP-O Coproporphyrin oxidase
8
Xenobiotics target in Heme biosynthetic pathway at 3 sides Xenobiotics
++
glycine ALA-S
ALA SUCC 8-CP
-
-CPOX
6-CP 5-CP 4-CP 2-CP
7-CP
Heme
Laboratoire Philippe Auguste
9
Xenobiotic compounds
Laboratoire Philippe Auguste
10
Aluminium (Al)
Laboratoire Philippe Auguste
11
Arsenic (As)
Laboratoire Philippe Auguste
12
LEAD TARGETS 3 ENZYMES OF THE HEME BIOSYNTHETIC PATHWAY
Laboratoire Philippe Auguste
13
HEME
1334 49911
Kreb Cycle
Protoporphyrinogen
1333
SuccinylCoA
Pb
23137
Glycine
5-Amino-levulinate (ALA) Coproporphyrinogen
42124 41137 4318
Porphobilinogen
Uroporphyrinogen
42175
Laboratoire Philippe Auguste 14
PORPHYRIN BIOSYNTHETIC PATHWAY PROVIDES 3 MARKERS OF LEAD TOXICITY
Targeted Enzymes
Biological Markers
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
Laboratoire Philippe Auguste
15
Lead (Pb)
Laboratoire Philippe Auguste
16
UROPORPHYRIN DECARBOXYLASE POSSESSES 4 DISTINCT SITES OF DECARBOXYLATION
Hg inhibits only the 4th site of decarboxylation generating accumulation 5CXP
UROPORPHYRIN DECARBOXYLASE (MW 80000)
1st site -CO2 2nd site -CO2 3rd site -CO2 4th site -CO2
8-carboxy-P or Uro-P
7-carboxy-P
6-carboxy-P
5-carboxy-P
4-carboxy-P or Copro-P
Laboratoire Philippe Auguste
17
Mercury targets CPOX and UROD in Heme biosynthetic pathway Hg
UROD
8-CP 7-CP 6-CP
CPOX
5-CP
4-CP
2-CP
Heme
CPOX4
KICP
Promotion in presence of Hg
James S. Woods et al.. Texicology Letter 2005….
Laboratoire Philippe Auguste
18
Mercury (Hg)
Laboratoire Philippe Auguste
19
WO JA
Age = 3 Urinary Hg = 0.048 µg/l = 0.065 µg/gCr
Moderate Hg toxic effect
PCP/URO = 1 5CXP >7CXP COP increased
Laboratoire Philippe Auguste
20
Urinary Hg = 0.41 µg/l = 0.235 µg/gCr
Moderate Hg toxic effect
PCP/URO = 1 5CXP > 7CXP association? Uncoupled COP
Laboratoire Philippe Auguste
21
NI AN
Age = 5 Urinary Hg = 0.11 µg/gCr
Noticeable Hg toxic effect
PCP = 1. 3 URO 5CXP > 7CXP = 1.5 increased COP
Laboratoire Philippe Auguste
22
WE RI
Urinary Hg = 0.337 µg/l = 0.272 µg/gCr Age = 4
High and exclusive Hg toxic effect
High PCP/URO > 4 PCP/5CXP > 5 5CXP/7CXP > 2, repression URO level and Por Synth Rate ?
Laboratoire Philippe Auguste
23
SO KE
Age = 3 Urinary Hg = 0.058 µg/gCr
High Hg toxic effect
PCP twice higher URO 5CXP > 7CXP high COP xenobiotics ? lead ?
Laboratoire Philippe Auguste
24
SA SA
Urinary Hg = 1.06 µg/gCr
Estonian child with MENTAL RETARDATION probably no up regulated porphyrin synthesis but polyhalogenated uro-D inhibition, and increased Hg specific porphyrins
Age = 2
Laboratoire Philippe Auguste
25
ZH SA
Age = 2 Urinary Hg = 0.312 µg/gCr
High Hg toxic effect Xenobiotics Up-regulation porphyrin synthesis PCP twice higher URO 5CXP > 7CXP high COP
Laboratoire Philippe Auguste
26
brothers
Laboratoire Philippe Auguste
27
Twin Brothers
Laboratoire Philippe Auguste
28
Parents
Laboratoire Philippe Auguste
29
High Hg Toxicity
Laboratoire Philippe Auguste
30
Pre-Chelation
Post-Chelation
04/07/07
17/12/07
precopro precopro
Laboratoire Philippe Auguste
31
Pre-Chelation
Post-Chelation
14/11/07
02/01/08
Laboratoire Philippe Auguste
32
25/04/07
Pre-Chelation
29/11/07
Post-Chelation
Laboratoire Philippe Auguste
33
Pre-Chelation
Post-Chelation
08/06/07
12/11/07
Laboratoire Philippe Auguste
34
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
35
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
36
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
37
Pre-Chelation
Post-Chelation
10/07
12/07
Laboratoire Philippe Auguste
38
Pre-Chelation
Post-Chelation
26/07/07
02/11/07
Laboratoire Philippe Auguste
39
15/06/07
Pre-Chelation
12/11/07
Post-Chelation
Laboratoire Philippe Auguste
40
01/06/07
Pre-Chelation
13/11/07
Post-Chelation
Laboratoire Philippe Auguste
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22/08/07
Pre-Chelation
18/12/07
Post-Chelation
Laboratoire Philippe Auguste
42
Pre-Chelation
Post-Chelation
Laboratoire Philippe Auguste
43
Porphyrinuria in childhood autistic disorder, Implication for environmental Toxicity
Toxicology and Applied Pharmacology (June, 2006)
Nataf R, Skorupka C, Amet L, Lam A, Springbett, Lathe R,
Laboratoire Philippe Auguste
44
269 Study Subjects (2002-2004)
Condition/diagnosis Allergy Asperger Attention deficit Autism (autistic disorder) Autism+epilepsy Cerebral palsy Epilepsy Hyperactivity MR+ epilepsy PDD-NOS Psychomotor retardation Rett Control group TOTAL
Laboratoire Philippe Auguste
M 5 10 2 79 7 6 2 27 1 51 1 0 7 198
F 3 1 7 27 2 6 0 2 1 12 3 2 5 71
Mean Total age (yr) 8 7,3 11 10 9 9,4 106 6,4 9 9,3 12 8,3 2 10 29 9,1 2 6 63 6,6 4 7,3 2 2,5 12 10,3 269 7,4
M/F 1,67 10 0,29 2,9 3,5 1 na 13,5 1 4,3 0,33 0 1,4 2,8
% total 3 4,1 3,3 39 3,3 4,4 0,7 10,7 0,7 23,4 1,5 0,7 4,4
% ASD group 5,8 55,5 ASD= 71% of total sample (M/F= 3,34)
33
1
45
Coproporphyrin (micromol/mol CRT) 10 20 30 40 50 60 70 80 0 ALLERGY ASPERGER
Laboratoire Philippe Auguste
ATTENTION DEFICIT
AUTISM
AUTISM + EPILEPSY
CEREBRAL PALSY
EPILEPSY
n=2
HYPERACTIVITY
MR + EPILEPSY
n=2
PDD-NOS
PSYCHOMOTOR RET
n=4
RETT
Coproporphyrin levels in urines of children with neurodevelopmental and related disorders
n=2
CONTROL GouRP
CTRL 2 x SD
CTRL MEAN
46
Elevated urinary Coproporphyrin (COPRO) levels in ASD expressed as absolute values normalised to creatinine (left) or as an internal ratio with uroporphyrin (URO) (right)
40
25
Coproporphyrin )
30
20
20
COPRO/URO ratio
AUT+EPI CTRL
15
10
10
5
AUT+EPI
0 AUT ASP
Laboratoire Philippe Auguste
CTRL
0 AUT ASP
47
Precoproporphyrin and pentacarboxy porphyrin: Markers of heavy metal toxicity
8
Control
8
ASP
8
AUT
8
AUT +EPI
7
7
6
6
6
6
(1.28)
5
5
4
(1.09)
4
4
4
3
3
2
(0.62)
2
(0.56)
2
2
1
1
0 0 2
0
0
0
2
0
0
2
4
0
2
4
uroporphyrin
Laboratoire Philippe Auguste
Precoproporphyrin plotted against baseline uroporphyrin : the ratio is independent of age-related creatinine variation
48
Spectrum of mean porphyrin excess, expressed as a ratio of control group (CTL) value for the different porphyrin subtypes
5
4 Porphyrin ratios to control group values
UROP
3
7CXP 6CXP 5CXP
2
COP PRECOP
1
0
ASP
Laboratoire Philippe Auguste
PDD-NOS
AUT
AUT+EPI
CTL
49
Neopterin Marker of Neuroinflammation
Neuroinflammation in Autism
x +
y
biopte rin
Oxidative Stress
Laboratoire Philippe Auguste
50
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.
Laboratoire Philippe Auguste
51
URINARY NEOPTERIN & BIOPTERIN
Neopterin (Autist)
Neopterin (control)
Biopterine (control)
Biopterine (Autist)
Laboratoire Philippe Auguste
52
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 Augusterelease reaktive oxygen species (ROS) and tumor necrosis factor-a (THF-a). Laboratoire Philippe also 53
Neopterin
Laboratoire Philippe Auguste
54
Laboratoire Philippe Auguste
55
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.
Laboratoire Philippe Auguste
56
Urinary Neopterine in Autism in 110 Children (Age between 2-11 years old)
nmole/mmole Cr 600 500 Neopterine 400 300 200 100 Autism
515 ±207
Laboratoire Philippe Auguste
n=159
n=32 Control
220±126
57
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
58
Pre-Chelation
Post-Chelation
10/07
12/07
Laboratoire Philippe Auguste
59
Pre-Chelation
Post-Chelation
Laboratoire Philippe Auguste
60
Oxidative Stress in Autism
Laboratoire Philippe Auguste
61
Is oxidative stress increased in Autism ?
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.
Laboratoire Philippe Auguste 62
Laboratoire Philippe Auguste
63
Laboratoire Philippe Auguste
64
8-oxo-deoxyguanosine (8OHdG)
Laboratoire Philippe Auguste 65
°OH
°O
8-oxo-guanosine (8-OG)
Laboratoire Philippe Auguste 66
8-oxo-deoxyguanosine (8OHdG)
8-oxo-guanosine (8-OHG)
Laboratoire Philippe Auguste
67
Main pathologies related to 8-oxo-DeoxyGuanosine increase
aging
Cancer
DNA oxidative damage marker DNA oxidative damage marker
to monitoroxidative stress onitor oxidative stress to m
8-oxo-deoxyguanosine
Cardio-vascular diseases
Intoxication
Laboratoire Philippe Auguste
68
Main pathologies related to 8-oxo-Guanosine increased
brain degenrative diseases Alzheimer Disease
Lateral Amyotrophic Sclerosis
RNA oxidative damage marker to asses oxidative stress
8-oxo-guanosine
Senile Dementia
Parkinson Disease
Laboratoire Philippe Auguste
69
RNA oxidation is an early and prominent feature of main brain neurodegenerative diseases AD PD Senile Dementia with lewy bodies ALS MSA-PD In AD cytoplasmic RNA oxidation by free radicals released by mitochondria is an early event of perikaryon degeneration precessing specific neurofibrillar tangles .
Laboratoire Philippe Auguste
70
Mitochondria failure Mitochondria failure MtDNA deletion MtDNA deletion H22O22 released in cytoplasm H O released in cytoplasm cytoplasmic iron -> highly reactive oxygen species cytoplasmic iron -> highly reactive oxygen species All RNA species (r,t,m) oxidation All RNA species (r,t,m) oxidation Protein synthesis impairment Protein synthesis impairment Neurodegenrative specific stigmats Neurodegenrative specific stigmats
Is cytoplasmic RNA oxidation a mitochondria’s failure consequence ?
Laboratoire Philippe Auguste 71
DNA and RNA Oxidative Markers preliminary results in 110 Children (Age between 2-11 years old)
nmole/gCr 100
87±33
Autism
43±27
Control 50
35±13
Autism
Control
29 ±1 1
8OHG
Laboratoire Philippe Auguste
8OHdG
72
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
73
Environment Intoxication?
Structure Modification
Inflammation
Function Loss
Stress Oxydant
Genetic
Pathogenic Interactions in Autism??
Laboratoire Philippe Auguste 74
Using Urinary Porphyrin, Neopterin and 8OHdG/8OHG Profile In Chelation Therapy
Laboratoire Philippe Auguste
75
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.
Laboratoire Philippe Auguste
76
5-Carboxyporphyrine
45 40 35 30 nmole/gCr 25 20 15 10 5 0 0
5-carboxyporphyrin
14 12 10 nmol/gCr 8 6 4 2 0
Pre-chelation 1
Post-chelation 2
3
PrePostchelation chelation
n=190 age = 1-12 years old
Laboratoire Philippe Auguste 77
Precoproporphyrin
80 70 60 nmole/gcr 50
nmol/gCr 45 40 35 30 25 20 15 10 5 0
Precoproporphyrin
40 30 20 10 0 0 Pre-chelation 1 Post-chelation 2 3
Pre-chelation
Post-chelation
n=190 age = 1-12 years old
Laboratoire Philippe Auguste
78
1000 900 800 700 600
Coproporphyrin
600 500 400 300 200 100 0
Coproporphyrin
nmole/gCr
500 400 300 200 100 0
Pre-chelation
Post-chelation
Pre-chelation
Post-chelation
n=190 age = 1-12 years old
Laboratoire Philippe Auguste
79
As the same time decrease inflammation, as shown by reduction in macrophage activation issued Neopterins.
neopterines
1400 1200 1000 nmol/gCr 800 600 400 200 0 pre-chelation post-chelation
n=39 age = 3-10 years old 700 600 500 nmol/gCr 400 300 200 100 0
neopterins
pre-chelation
post-chelation
Laboratoire Philippe Auguste
80
And decreaseoxidative stress , inside nucleus and through cytoplasm, as shown by 8-oxo-Deoxyguanosine (8OHdG) and 8-oxo-Guanosine(8OHG) reduction.
Oxidative Stress
160 140 120 100 nmole/gCr 80 60 40 20 0 pre-chelation post-chelation pre-chelation post-chelation
nmol/gCr 90 80
Oxidative Stress
n=23 age 2 -12 years old
8OHG
8OHdG
8OHG
70 60 50 40 30 20 10 0
8OHdG
Pre-chelation Post-chelation
Pre-chelation Post-chelation
Laboratoire Philippe Auguste
81
Laboratoire Philippe Auguste
82
Improvement in biological indicators in Autism: properly define clinical improvement
Robert Nataf M.D.
Laboratoire Philippe Auguste 2 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 : contact@labbio.net
Laboratoire Philippe Auguste 1
Nervous system
Malnutrition Neuro-inflammation
Environment
Toxins Oxidative Stress
Autism
GI system
Genomes
Immune system
Detoxification system
Laboratoire Philippe Auguste 2
Biochemical Abnormalities in Autism
• Elevated toxic metals and Xenobiotics • Immune Disorder • Severe oxidative stress & damage
Laboratoire Philippe Auguste
3
Urinary Porphyrins: A Biomarker of Environmental Toxicity
Laboratoire Philippe Auguste
4
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.
Laboratoire Philippe Auguste
5
PORPHYRINS SYNTHESIS
8 enzymes ensures in mitochondrion and cytoplasm, the synthesis of porphyrins: It may be divided into 3 steps : • reaction of 2 simple molecules, glycine (from the general amino acid pool) and succinyl-CoA (from the tricarboxylic acid cycle) to Porphobilinogen. • Decarboxylation reaction from Uroporphyrin (8-carboxy) to Coproporphyrin (4-carboxy).
6
Laboratoire Philippe Auguste
3. The synthesis of heme from 2-carboxyporphyrin through two
M.W. 830
M.W. 786
M.W. 742
M.W. 566
Heme biosynthetic pathway
Laboratoire Philippe Auguste
1 = δ-aminolevulinicacid (ALA) synthetase 2 = ALA dehydratase 3 = uroporphyrinogen I synthetase PBGD 4 = uroporphyrinogen III cosynthetase 5 = UROD (Uroporphyrinogen decarboxylase) 6 = COPOX (Coproporphyrinogen oxidase) 7 = Protoporphyrinogen oxidase 8 = Ferrochelatase
M.W. 698
7
Polychorinated Biphenyl (PCB) Arsenic (As) Aluminium (Al)
UroP 7cxP
Mercury (Hg)
5 cxP PcP CoP
Lead (Pb)
CoP
Laboratoire Philippe Auguste
URO-D = Uroporphyrin Decarboxylase
COP-O Coproporphyrin oxidase
8
Xenobiotics target in Heme biosynthetic pathway at 3 sides Xenobiotics
++
glycine ALA-S
ALA SUCC 8-CP
-
-CPOX
6-CP 5-CP 4-CP 2-CP
7-CP
Heme
Laboratoire Philippe Auguste
9
Xenobiotic compounds
Laboratoire Philippe Auguste
10
Aluminium (Al)
Laboratoire Philippe Auguste
11
Arsenic (As)
Laboratoire Philippe Auguste
12
LEAD TARGETS 3 ENZYMES OF THE HEME BIOSYNTHETIC PATHWAY
Laboratoire Philippe Auguste
13
HEME
1334 49911
Kreb Cycle
Protoporphyrinogen
1333
SuccinylCoA
Pb
23137
Glycine
5-Amino-levulinate (ALA) Coproporphyrinogen
42124 41137 4318
Porphobilinogen
Uroporphyrinogen
42175
Laboratoire Philippe Auguste 14
PORPHYRIN BIOSYNTHETIC PATHWAY PROVIDES 3 MARKERS OF LEAD TOXICITY
Targeted Enzymes
Biological Markers
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
Laboratoire Philippe Auguste
15
Lead (Pb)
Laboratoire Philippe Auguste
16
UROPORPHYRIN DECARBOXYLASE POSSESSES 4 DISTINCT SITES OF DECARBOXYLATION
Hg inhibits only the 4th site of decarboxylation generating accumulation 5CXP
UROPORPHYRIN DECARBOXYLASE (MW 80000)
1st site -CO2 2nd site -CO2 3rd site -CO2 4th site -CO2
8-carboxy-P or Uro-P
7-carboxy-P
6-carboxy-P
5-carboxy-P
4-carboxy-P or Copro-P
Laboratoire Philippe Auguste
17
Mercury targets CPOX and UROD in Heme biosynthetic pathway Hg
UROD
8-CP 7-CP 6-CP
CPOX
5-CP
4-CP
2-CP
Heme
CPOX4
KICP
Promotion in presence of Hg
James S. Woods et al.. Texicology Letter 2005….
Laboratoire Philippe Auguste
18
Mercury (Hg)
Laboratoire Philippe Auguste
19
WO JA
Age = 3 Urinary Hg = 0.048 µg/l = 0.065 µg/gCr
Moderate Hg toxic effect
PCP/URO = 1 5CXP >7CXP COP increased
Laboratoire Philippe Auguste
20
Urinary Hg = 0.41 µg/l = 0.235 µg/gCr
Moderate Hg toxic effect
PCP/URO = 1 5CXP > 7CXP association? Uncoupled COP
Laboratoire Philippe Auguste
21
NI AN
Age = 5 Urinary Hg = 0.11 µg/gCr
Noticeable Hg toxic effect
PCP = 1. 3 URO 5CXP > 7CXP = 1.5 increased COP
Laboratoire Philippe Auguste
22
WE RI
Urinary Hg = 0.337 µg/l = 0.272 µg/gCr Age = 4
High and exclusive Hg toxic effect
High PCP/URO > 4 PCP/5CXP > 5 5CXP/7CXP > 2, repression URO level and Por Synth Rate ?
Laboratoire Philippe Auguste
23
SO KE
Age = 3 Urinary Hg = 0.058 µg/gCr
High Hg toxic effect
PCP twice higher URO 5CXP > 7CXP high COP xenobiotics ? lead ?
Laboratoire Philippe Auguste
24
SA SA
Urinary Hg = 1.06 µg/gCr
Estonian child with MENTAL RETARDATION probably no up regulated porphyrin synthesis but polyhalogenated uro-D inhibition, and increased Hg specific porphyrins
Age = 2
Laboratoire Philippe Auguste
25
ZH SA
Age = 2 Urinary Hg = 0.312 µg/gCr
High Hg toxic effect Xenobiotics Up-regulation porphyrin synthesis PCP twice higher URO 5CXP > 7CXP high COP
Laboratoire Philippe Auguste
26
brothers
Laboratoire Philippe Auguste
27
Twin Brothers
Laboratoire Philippe Auguste
28
Parents
Laboratoire Philippe Auguste
29
High Hg Toxicity
Laboratoire Philippe Auguste
30
Pre-Chelation
Post-Chelation
04/07/07
17/12/07
precopro precopro
Laboratoire Philippe Auguste
31
Pre-Chelation
Post-Chelation
14/11/07
02/01/08
Laboratoire Philippe Auguste
32
25/04/07
Pre-Chelation
29/11/07
Post-Chelation
Laboratoire Philippe Auguste
33
Pre-Chelation
Post-Chelation
08/06/07
12/11/07
Laboratoire Philippe Auguste
34
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
35
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
36
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
37
Pre-Chelation
Post-Chelation
10/07
12/07
Laboratoire Philippe Auguste
38
Pre-Chelation
Post-Chelation
26/07/07
02/11/07
Laboratoire Philippe Auguste
39
15/06/07
Pre-Chelation
12/11/07
Post-Chelation
Laboratoire Philippe Auguste
40
01/06/07
Pre-Chelation
13/11/07
Post-Chelation
Laboratoire Philippe Auguste
41
22/08/07
Pre-Chelation
18/12/07
Post-Chelation
Laboratoire Philippe Auguste
42
Pre-Chelation
Post-Chelation
Laboratoire Philippe Auguste
43
Porphyrinuria in childhood autistic disorder, Implication for environmental Toxicity
Toxicology and Applied Pharmacology (June, 2006)
Nataf R, Skorupka C, Amet L, Lam A, Springbett, Lathe R,
Laboratoire Philippe Auguste
44
269 Study Subjects (2002-2004)
Condition/diagnosis Allergy Asperger Attention deficit Autism (autistic disorder) Autism+epilepsy Cerebral palsy Epilepsy Hyperactivity MR+ epilepsy PDD-NOS Psychomotor retardation Rett Control group TOTAL
Laboratoire Philippe Auguste
M 5 10 2 79 7 6 2 27 1 51 1 0 7 198
F 3 1 7 27 2 6 0 2 1 12 3 2 5 71
Mean Total age (yr) 8 7,3 11 10 9 9,4 106 6,4 9 9,3 12 8,3 2 10 29 9,1 2 6 63 6,6 4 7,3 2 2,5 12 10,3 269 7,4
M/F 1,67 10 0,29 2,9 3,5 1 na 13,5 1 4,3 0,33 0 1,4 2,8
% total 3 4,1 3,3 39 3,3 4,4 0,7 10,7 0,7 23,4 1,5 0,7 4,4
% ASD group 5,8 55,5 ASD= 71% of total sample (M/F= 3,34)
33
1
45
Coproporphyrin (micromol/mol CRT) 10 20 30 40 50 60 70 80 0 ALLERGY ASPERGER
Laboratoire Philippe Auguste
ATTENTION DEFICIT
AUTISM
AUTISM + EPILEPSY
CEREBRAL PALSY
EPILEPSY
n=2
HYPERACTIVITY
MR + EPILEPSY
n=2
PDD-NOS
PSYCHOMOTOR RET
n=4
RETT
Coproporphyrin levels in urines of children with neurodevelopmental and related disorders
n=2
CONTROL GouRP
CTRL 2 x SD
CTRL MEAN
46
Elevated urinary Coproporphyrin (COPRO) levels in ASD expressed as absolute values normalised to creatinine (left) or as an internal ratio with uroporphyrin (URO) (right)
40
25
Coproporphyrin )
30
20
20
COPRO/URO ratio
AUT+EPI CTRL
15
10
10
5
AUT+EPI
0 AUT ASP
Laboratoire Philippe Auguste
CTRL
0 AUT ASP
47
Precoproporphyrin and pentacarboxy porphyrin: Markers of heavy metal toxicity
8
Control
8
ASP
8
AUT
8
AUT +EPI
7
7
6
6
6
6
(1.28)
5
5
4
(1.09)
4
4
4
3
3
2
(0.62)
2
(0.56)
2
2
1
1
0 0 2
0
0
0
2
0
0
2
4
0
2
4
uroporphyrin
Laboratoire Philippe Auguste
Precoproporphyrin plotted against baseline uroporphyrin : the ratio is independent of age-related creatinine variation
48
Spectrum of mean porphyrin excess, expressed as a ratio of control group (CTL) value for the different porphyrin subtypes
5
4 Porphyrin ratios to control group values
UROP
3
7CXP 6CXP 5CXP
2
COP PRECOP
1
0
ASP
Laboratoire Philippe Auguste
PDD-NOS
AUT
AUT+EPI
CTL
49
Neopterin Marker of Neuroinflammation
Neuroinflammation in Autism
x +
y
biopte rin
Oxidative Stress
Laboratoire Philippe Auguste
50
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.
Laboratoire Philippe Auguste
51
URINARY NEOPTERIN & BIOPTERIN
Neopterin (Autist)
Neopterin (control)
Biopterine (control)
Biopterine (Autist)
Laboratoire Philippe Auguste
52
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 Augusterelease reaktive oxygen species (ROS) and tumor necrosis factor-a (THF-a). Laboratoire Philippe also 53
Neopterin
Laboratoire Philippe Auguste
54
Laboratoire Philippe Auguste
55
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.
Laboratoire Philippe Auguste
56
Urinary Neopterine in Autism in 110 Children (Age between 2-11 years old)
nmole/mmole Cr 600 500 Neopterine 400 300 200 100 Autism
515 ±207
Laboratoire Philippe Auguste
n=159
n=32 Control
220±126
57
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
58
Pre-Chelation
Post-Chelation
10/07
12/07
Laboratoire Philippe Auguste
59
Pre-Chelation
Post-Chelation
Laboratoire Philippe Auguste
60
Oxidative Stress in Autism
Laboratoire Philippe Auguste
61
Is oxidative stress increased in Autism ?
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.
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Laboratoire Philippe Auguste
63
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64
8-oxo-deoxyguanosine (8OHdG)
Laboratoire Philippe Auguste 65
°OH
°O
8-oxo-guanosine (8-OG)
Laboratoire Philippe Auguste 66
8-oxo-deoxyguanosine (8OHdG)
8-oxo-guanosine (8-OHG)
Laboratoire Philippe Auguste
67
Main pathologies related to 8-oxo-DeoxyGuanosine increase
aging
Cancer
DNA oxidative damage marker DNA oxidative damage marker
to monitoroxidative stress onitor oxidative stress to m
8-oxo-deoxyguanosine
Cardio-vascular diseases
Intoxication
Laboratoire Philippe Auguste
68
Main pathologies related to 8-oxo-Guanosine increased
brain degenrative diseases Alzheimer Disease
Lateral Amyotrophic Sclerosis
RNA oxidative damage marker to asses oxidative stress
8-oxo-guanosine
Senile Dementia
Parkinson Disease
Laboratoire Philippe Auguste
69
RNA oxidation is an early and prominent feature of main brain neurodegenerative diseases AD PD Senile Dementia with lewy bodies ALS MSA-PD In AD cytoplasmic RNA oxidation by free radicals released by mitochondria is an early event of perikaryon degeneration precessing specific neurofibrillar tangles .
Laboratoire Philippe Auguste
70
Mitochondria failure Mitochondria failure MtDNA deletion MtDNA deletion H22O22 released in cytoplasm H O released in cytoplasm cytoplasmic iron -> highly reactive oxygen species cytoplasmic iron -> highly reactive oxygen species All RNA species (r,t,m) oxidation All RNA species (r,t,m) oxidation Protein synthesis impairment Protein synthesis impairment Neurodegenrative specific stigmats Neurodegenrative specific stigmats
Is cytoplasmic RNA oxidation a mitochondria’s failure consequence ?
Laboratoire Philippe Auguste 71
DNA and RNA Oxidative Markers preliminary results in 110 Children (Age between 2-11 years old)
nmole/gCr 100
87±33
Autism
43±27
Control 50
35±13
Autism
Control
29 ±1 1
8OHG
Laboratoire Philippe Auguste
8OHdG
72
Pre-Chelation
Post-Chelation
20/06/07
12/10/07
Laboratoire Philippe Auguste
73
Environment Intoxication?
Structure Modification
Inflammation
Function Loss
Stress Oxydant
Genetic
Pathogenic Interactions in Autism??
Laboratoire Philippe Auguste 74
Using Urinary Porphyrin, Neopterin and 8OHdG/8OHG Profile In Chelation Therapy
Laboratoire Philippe Auguste
75
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.
Laboratoire Philippe Auguste
76
5-Carboxyporphyrine
45 40 35 30 nmole/gCr 25 20 15 10 5 0 0
5-carboxyporphyrin
14 12 10 nmol/gCr 8 6 4 2 0
Pre-chelation 1
Post-chelation 2
3
PrePostchelation chelation
n=190 age = 1-12 years old
Laboratoire Philippe Auguste 77
Precoproporphyrin
80 70 60 nmole/gcr 50
nmol/gCr 45 40 35 30 25 20 15 10 5 0
Precoproporphyrin
40 30 20 10 0 0 Pre-chelation 1 Post-chelation 2 3
Pre-chelation
Post-chelation
n=190 age = 1-12 years old
Laboratoire Philippe Auguste
78
1000 900 800 700 600
Coproporphyrin
600 500 400 300 200 100 0
Coproporphyrin
nmole/gCr
500 400 300 200 100 0
Pre-chelation
Post-chelation
Pre-chelation
Post-chelation
n=190 age = 1-12 years old
Laboratoire Philippe Auguste
79
As the same time decrease inflammation, as shown by reduction in macrophage activation issued Neopterins.
neopterines
1400 1200 1000 nmol/gCr 800 600 400 200 0 pre-chelation post-chelation
n=39 age = 3-10 years old 700 600 500 nmol/gCr 400 300 200 100 0
neopterins
pre-chelation
post-chelation
Laboratoire Philippe Auguste
80
And decreaseoxidative stress , inside nucleus and through cytoplasm, as shown by 8-oxo-Deoxyguanosine (8OHdG) and 8-oxo-Guanosine(8OHG) reduction.
Oxidative Stress
160 140 120 100 nmole/gCr 80 60 40 20 0 pre-chelation post-chelation pre-chelation post-chelation
nmol/gCr 90 80
Oxidative Stress
n=23 age 2 -12 years old
8OHG
8OHdG
8OHG
70 60 50 40 30 20 10 0
8OHdG
Pre-chelation Post-chelation
Pre-chelation Post-chelation
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81
Laboratoire Philippe Auguste
82
