Improvement in biological indicators in Autism: properly define clinical improvement Robert Nataf M.D. • Laboratoire Philippe Auguste • Avenue Philippe Auguste • 75011 Paris France • http://www.labbio.net • Tel: (33)22.214.171.124.00 Fax: (33)1.43.79.00.27 • Email : firstname.lastname@example.org
Nervous system Malnutrition Neuro-inflammation Environment Autism Toxins Oxidative Stress GI system Immune system Genomes Detoxification system
Biochemical Abnormalities in Autism • Elevated toxic metals and Xenobiotics • Immune Disorder • Severe oxidative stress & damage
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.
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.
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
Toxic sensitivity of different porphyrins Polychorinated Biphenyl (PCB) UroP Arsenic (As) 7cxP Aluminium (Al) Mercury (Hg) 5 cxP PcP CoP Lead (Pb) CoP COP-O COP-OX URO-D URO-D URO-D Heme URO-D = Uroporphyrin Decarboxylase COP-O Coproporphyrin oxidase
Xenobiotics target in Heme biosynthetic pathway at 3 sides Xenobiotics - + + - - glycine - ALA-S CPOX 8-CP 6-CP 7-CP 5-CP 4-CP 2-CP ALA SUCC Heme
LEAD TARGETS 3 ENZYMES OF THE HEME BIOSYNTHETIC PATHWAY
Kreb Cycle HEME 49911 1334 Succinyl-CoA Protoporphyrinogen Pb 23137 Glycine 1333 5-Amino-levulinate (ALA) Coproporphyrinogen 42124 41137 4318 Porphobilinogen Uroporphyrinogen 42175
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
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
Mercury targets CPOX and UROD in Heme biosynthetic pathway Hg UROD CPOX 8-CP 6-CP 7-CP 5-CP 4-CP 2-CP Heme CPOX4 KICP Promotion in presence of Hg James S. Woods et al.. Texicology Letter 2005….
WO JA Age = 3 Moderate Hg toxic effect Urinary Hg = 0.048 µg/l = 0.065 µg/gCr PCP/URO = 1 5CXP >7CXP COP increased
Moderate Hg toxic effect Urinary Hg = 0.41 µg/l = 0.235 µg/gCr PCP/URO = 1 5CXP > 7CXP association? Uncoupled COP
NI AN Noticeable Hg toxic effect Age = 5 Urinary Hg = 0.11 µg/gCr PCP = 1. 3 URO 5CXP > 7CXP = 1.5 increased COP
WE RI Age = 4 Urinary Hg = 0.337 µg/l = 0.272 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 ?
SO KE Age = 3 Urinary Hg = 0.058 µg/gCr High Hg toxic effect PCP twice higher URO 5CXP > 7CXP high COP xenobiotics ? lead ?
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
ZH SA High Hg toxic effect Xenobiotics Age = 2 Urinary Hg = 0.312 µg/gCr Up-regulation porphyrin synthesis PCP twice higher URO 5CXP > 7CXP high COP
Post-Chelation Pre-Chelation 04/07/07 17/12/07 precopro precopro
Pre-Chelation Post-Chelation 14/11/07 02/01/08
25/04/07 29/11/07 Post-Chelation Pre-Chelation
Post-Chelation Pre-Chelation 12/11/07 08/06/07
Pre-Chelation Post-Chelation 20/06/07 12/10/07
Post-Chelation Pre-Chelation 20/06/07 12/10/07
Post-Chelation Pre-Chelation 20/06/07 12/10/07
Post-Chelation Pre-Chelation 10/07 12/07
Post-Chelation Pre-Chelation 26/07/07 02/11/07
12/11/07 15/06/07 Post-Chelation Pre-Chelation
01/06/07 13/11/07 Post-Chelation Pre-Chelation
22/08/07 18/12/07 Post-Chelation Pre-Chelation
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,
Coproporphyrin levels in urines of children with neurodevelopmental and related disorders n=2 n=2 n=2 n=4 CTRL 2 x SD CTRL MEAN
Elevated urinary Coproporphyrin (COPRO) levels in ASD expressed as absolute values normalised to creatinine (left) or as an internal ratio with uroporphyrin (URO) (right)
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
Spectrum of mean porphyrin excess, expressed as a ratio of control group (CTL) value for the different porphyrin subtypes
Neopterin Marker of Neuroinflammation Neuroinflammation in Autism