1. Improvement in biological indicators in Autism: properly define clinical improvement
Robert Nataf M.D.
Laboratoire Philippe Auguste
Avenue Philippe Auguste
Paris France
Tel: (33) Fax: (33)
Laboratoire Philippe Auguste

2. Detoxification system
Nervous system
Malnutrition
Neuro-inflammation
Environment
Autism
Toxins
Oxidative Stress
GI system
Immune system
Genomes
Detoxification system
Laboratoire Philippe Auguste

3. Biochemical Abnormalities in Autism
Elevated toxic metals and Xenobiotics
Immune Disorder
Severe oxidative stress & damage
Laboratoire Philippe Auguste

4. Urinary Porphyrins: A Biomarker of Environmental Toxicity
Laboratoire Philippe Auguste

5. 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

6. 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).
3. The synthesis of heme from 2-carboxyporphyrin through two last biosynthetic pathway enzymes.
Laboratoire Philippe Auguste

7. Heme biosynthetic pathway
M.W. 742
M.W. 830
M.W. 786
M.W. 566
M.W. 698
1 = d-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
Heme biosynthetic pathway
Laboratoire Philippe Auguste

8. Toxic sensitivity of different porphyrins
Polychorinated Biphenyl (PCB) UroP
Arsenic (As) cxP
Aluminium (Al)
Mercury (Hg) cxP
PcP
CoP
Lead (Pb) CoP
COP-O
COP-OX
URO-D
URO-D
URO-D
Heme
Laboratoire Philippe Auguste
URO-D = Uroporphyrin Decarboxylase
COP-O Coproporphyrin oxidase

9. Xenobiotics target in Heme biosynthetic pathway at 3 sides- + + - -
glycine -
ALA-S
CPOX
8-CP
6-CP
7-CP
5-CP
4-CP
2-CP
ALA
SUCC
Heme
Laboratoire Philippe Auguste

10. Xenobiotic compounds
Laboratoire Philippe Auguste

11. Aluminium (Al)
Laboratoire Philippe Auguste

12. Arsenic (As)
Laboratoire Philippe Auguste

13. 3 ENZYMES OF THE HEME BIOSYNTHETIC PATHWAY
LEAD TARGETS
3 ENZYMES OF THE HEME BIOSYNTHETIC PATHWAY
Laboratoire Philippe Auguste

14. 5-Amino-levulinate (ALA)
Kreb Cycle
HEME
49911
1334
Succinyl-CoA
Protoporphyrinogen Pb
23137
Glycine
1333
5-Amino-levulinate (ALA)
Coproporphyrinogen
42124
41137
4318
Porphobilinogen
Uroporphyrinogen
42175
Laboratoire Philippe Auguste

15. 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

16. Lead (Pb)
Laboratoire Philippe Auguste

17. 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
2nd site
3rd site
4th site
-CO2
-CO2
-CO2
-CO2
6-carboxy-P
5-carboxy-P
4-carboxy-P or Copro-P
8-carboxy-P or Uro-P
7-carboxy-P
Laboratoire Philippe Auguste

18. Mercury targets CPOX and UROD in Heme biosynthetic pathwayHg
UROD
CPOX
8-CP
7-CP
6-CP
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

19. Mercury (Hg)
Laboratoire Philippe Auguste

20. Moderate Hg toxic effect
WO JA
Age = 3
Moderate Hg toxic effect
Urinary Hg = µg/l
= µg/gCr
PCP/URO = 1
5CXP >7CXP
COP increased
Laboratoire Philippe Auguste

21. Moderate Hg toxic effect
Urinary Hg = µg/l
= µg/gCr
PCP/URO = 1
5CXP > 7CXP
association? Uncoupled COP
Laboratoire Philippe Auguste

22. Noticeable Hg toxic effect
NI AN
Noticeable Hg toxic effect
Age = 5
Urinary Hg = 0.11 µg/gCr
PCP = 1. 3 URO
5CXP > 7CXP = 1.5
increased COP
Laboratoire Philippe Auguste

23. High and exclusive Hg toxic effect
WE RI
Age = 4
Urinary Hg = µg/l
= µg/gCr
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

24. High Hg toxic effect SO KE PCP twice higher URO 5CXP > 7CXP
Age = 3
Urinary Hg = µg/gCr
High Hg toxic effect
PCP twice higher URO
5CXP > 7CXP
high COP
xenobiotics ?
lead ?
Laboratoire Philippe Auguste

25. Estonian child with MENTAL RETARDATION
SA SA
Estonian child with MENTAL RETARDATION
Urinary Hg = 1.06 µg/gCr
Age = 2
probably no up regulated porphyrin synthesis but polyhalogenated uro-D inhibition, and increased Hg specific porphyrins
Laboratoire Philippe Auguste

26. High Hg toxic effect Xenobiotics
ZH SA
High Hg toxic effect
Xenobiotics
Age = 2
Urinary Hg = µg/gCr
Up-regulation porphyrin synthesis
PCP twice higher URO
5CXP > 7CXP
high COP
Laboratoire Philippe Auguste

27. brothers
Laboratoire Philippe Auguste

28. Twin Brothers
Laboratoire Philippe Auguste

29. Parents
Laboratoire Philippe Auguste

30. High Hg Toxicity
Laboratoire Philippe Auguste

31. 04/07/07 17/12/07 precopro precopro Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

32. 14/11/07 02/01/08 Pre-Chelation Post-Chelation
Laboratoire Philippe Auguste

33. 25/04/07 29/11/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

34. 12/11/07 08/06/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

35. 20/06/07 12/10/07 Pre-Chelation Post-Chelation
Laboratoire Philippe Auguste

36. 20/06/07 12/10/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

37. 20/06/07 12/10/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

38. Post-Chelation
Pre-Chelation
10/07
12/07
Laboratoire Philippe Auguste

39. 26/07/07 02/11/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

40. 12/11/07 15/06/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

41. 01/06/07 13/11/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

42. 22/08/07 18/12/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

43. Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

44. 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

45. 269 Study Subjects ( )
Laboratoire Philippe Auguste

46. Coproporphyrin levels in urines of children with neurodevelopmental and related disorders
CTRL 2 x SD
CTRL MEAN
Laboratoire Philippe Auguste

47. Elevated urinary Coproporphyrin (COPRO) levels in ASD expressed as absolute values normalised to creatinine (left) or as an internal ratio with uroporphyrin (URO) (right)
Laboratoire Philippe Auguste

48. Precoproporphyrin and pentacarboxy porphyrin: Markers of heavy metal toxicity
Control
ASP
AUT
AUT +EPI
(1.28)
(1.09)
(0.62)
(0.56)
uroporphyrin
Precoproporphyrin plotted against baseline uroporphyrin : the ratio is independent of age-related creatinine variation
Laboratoire Philippe Auguste

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

50. Neopterin Marker of Neuroinflammation
Neuroinflammation in Autism
Laboratoire Philippe Auguste

51. 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

52. Laboratoire Philippe Auguste

53. 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).
Laboratoire Philippe Auguste

54. Neopterin
Laboratoire Philippe Auguste

55. Laboratoire Philippe Auguste

56. 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

57. Urinary Neopterine in Autism in 110 Children
(Age between 2-11 years old)
nmole/mmole Cr
600
500
400
Neopterine
n=159
300
200
100
n=32
Autism
Control
515 207
220126
Laboratoire Philippe Auguste

58. 20/06/07 12/10/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

59. Post-Chelation
Pre-Chelation
10/07
12/07
Laboratoire Philippe Auguste

60. Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

61. Oxidative Stress in Autism
Laboratoire Philippe Auguste

62. 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

63. Laboratoire Philippe Auguste

64. Laboratoire Philippe Auguste

65. 8-Oxo-Deoxyguanosine (8OHdG)
(DNA damage)
Laboratoire Philippe Auguste

66. Laboratoire Philippe Auguste

67. 8-oxo-deoxyguanosine (8OHdG) 8-oxo-guanosine (8-OHG)
Laboratoire Philippe Auguste

68. Main pathologies related to 8-oxo-DeoxyGuanosine increase
Laboratoire Philippe Auguste

69. Main pathologies related to 8-oxo-Guanosine increased
brain degenrative
diseases
Lateral Amyotrophic
Sclerosis
Alzheimer Disease
8-oxo-guanosine
RNA oxidative damage marker
to asses oxidative stress
Senile Dementia
Parkinson Disease
Laboratoire Philippe Auguste

70. Senile Dementia with lewy bodies
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

71. Is cytoplasmic RNA oxidation a mitochondria’s failure consequence ?
Laboratoire Philippe Auguste

72. DNA and RNA Oxidative Markers preliminary results in 110 Children
(Age between 2-11 years old)
nmole/gCr
8733
Autism
100
4327
Control 50
3513
29 11
Autism
Control
8OHdG
8OHG
Laboratoire Philippe Auguste

73. 20/06/07 12/10/07 Post-Chelation Pre-Chelation
Laboratoire Philippe Auguste

74. Environment Intoxication? Structure Modification
Inflammation
Stress Oxydant
Function Loss
Genetic
Pathogenic Interactions in Autism??
Laboratoire Philippe Auguste

75. Using Urinary Porphyrin, Neopterin and 8OHdG/8OHG Profile In Chelation Therapy
Laboratoire Philippe Auguste

76. 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

77. n=190 age = 1-12 years old Pre-chelation Post-chelation
Laboratoire Philippe Auguste

78. n=190 age = 1-12 years old Pre-chelation Post-chelation
Laboratoire Philippe Auguste

79. n=190 age = 1-12 years old Pre-chelation Post-chelation
Laboratoire Philippe Auguste

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

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

82. Thank you
Laboratoire Philippe Auguste