Looking%20Inside%20the%20Black%20Box:%20%20Understanding%20Analytical%20Approaches,%20Diagnostic%20Tools,%20and%20Detoxification%20Strategies%20for%20Mercury%20Intoxication - PowerPoint PPT Presentation

Title: Looking%20Inside%20the%20Black%20Box:%20%20Understanding%20Analytical%20Approaches,%20Diagnostic%20Tools,%20and%20Detoxification%20Strategies%20for%20Mercury%20Intoxication


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Looking Inside the Black Box Understanding
Analytical Approaches, Diagnostic Tools, and
Detoxification Strategies for Mercury Intoxication
Heretical Content
Christopher W. Shade, Ph.D. chris_at_quicksilverscien
tific.com Quicksilver Scientific, LLC Lafayette,
CO 80026 (303)263-6903
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Mercury and The Human Detoxification System

• What the key forms of Hg are
• How the Natural Detoxification System Works
• How the Detox System gets subverted leading to
  biochemical stress
• Mercury Analysis (Blood is not a dirty word)
• Intestinal Metals Detox System to repair and
  amplify the bodies Detoxification System and
  safely remove mercury

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Forms of mercury

• Hg0 Elemental Mercury
• The metal form both liquid and gas forms
• HgII Inorganic Mercury
• The salt, formed by oxidation of Hg0
• MeHg - Methylmercury
• Organomercurial, formed by bacterial synthesis
• EtHg - Ethylmercury
• Synthetic organomercurial antimicrobial

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Transport of mercury

• Hg0
• 80 uptake in lungs, crossed BBB, diffuses in to
  tissues moderate uptake from intestines
• HgII
• Very poor uptakes in intestines poor mobility
  does not cross BBB
• MeHg
• 95 uptake from intestines, good mobility,
  crosses BBB
• EtHg
• 100 absorbtion (inj), good mobility, crosses BBB

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Targets of mercury forms

• Brain/CNS
• Kidney
• Liver
• Heart
• Pituitary/Thyroid
• And thus all glandular

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The Heart of the Toxicity

1. Inappropriate Binding enzymes, redox proteins,
   membranes
2. Oxidative Damage and related Inflammation,
   including membrane damage (Parinanada) and
   apoptosis (cell death)

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The Heart of the Toxicity

• Thiol Binding and Redox Reaction
• Reduced sulfur groups, R-SH,
• Hg replaces proton and binds to sulfur
• R-SH Hg2 R-SHg H
• Enzymes use thiols to anchor functional metals
  (Zn, Ni, Cu, Fe)
• Bind and alter membrane or trigger membrane
  reorganization and consequent auto-oxidation
• Oxidize Thioredoxin (protein repair molecule)
• Deplete Glutathione system

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Inorganic Hg and Poisoning of the Extracellular
Matrix
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Mercury and The Human Detoxification System

• What the key forms of Hg are
• How the Natural Detoxification System Works
• How the Detox System gets subverted leading to
  biochemical stress
• Mercury Analysis (Blood is not a dirty word)
• Intestinal Metals Detox System to repair and
  amplify the bodies Detoxification System and
  safely remove mercury

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Defense Glutathione System Antioxidant,
Detoxification, Protein Repair

• Glutathione (GSH) - A thiolic tripeptide composed
  of glutamate, cysteine, and glycine

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Defense Glutathione System Antioxidant,
Detoxification, Protein Repair

• Synthetases (synthesize GSH from precursors)
• Transpeptidases (take apart and reassemble)
• Transferases (Phase II conjugation)
• Peroxidases (radical quenching)
• Reductases (repair after quenching)
• Redoxins (using GSH as reducing equivalent for
  protein repair)
• Glutathionylation protection of Proteins
• What to do? Quench the fire or protect the
  children?

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The Human Detoxification System

• Detoxification Phases I, II, III
• Phase I is an activation,
• Phase II is conjugation
• Phase III is transport (recently delineated
  control point)

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The Human Detoxification System

• Phase I - an oxidative activation, usually the
  Cytochrome P450 system
• Prepares toxin for conjugation in Phase II with
  GSH, Glucuronic acid, Sulfate, Gycine or other
  amino acid, Taurine, Methyl group
• Not needed for metals, but very important to have
  coupled to Phase II
• Creates Essentially Free-Radicals

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The Human Detoxification System

• Phase II conjugation makes toxin more water
  soluble and recognizable by transporters
• Glutathione S-Transferases (GST) responsible for
  GSH conjugation
• Low expression in people with high MeHg and with
  sensitivity (allergy) to Thimerosal (EthylHg)

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The Human Detoxification System

• Phase III is the transport out!
• Several transport proteins (cMOAT, OAT, MRP1,
  MRP2, GS-X)
• Organic Anion Transporters
• Same transporters for many pathways (glucuronide,
  sulfate, glycinate, GSH)
• In cells, liver, intestines, kidneys biggest in
  liver then intestines

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Mercury and The Human Detoxification System

• What the key forms of Hg are
• How the Natural Detoxification System Works
• How the Detox System gets subverted leading to
  biochemical stress
• Mercury Analysis (Blood is not a dirty word)
• Intestinal Metals Detox System to repair and
  amplify the bodies Detoxification System and
  safely remove mercury

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Breakdown of the defense system

• GSH deficiency
• Genetic (GCS polymorphisms, epigenetic
  dysfunction)
• Environmental (oxidative consumption or
  inflammation)
• GST problems
• Genetic (GST polymorphisms, epigenetic
  dysfunction)
• Environmental (Inflammatory cascade/ARE
  dysfunction)

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Disease and Gen Polymorphisms of GSH genes

• Hemolytic Anemia GST (Beutler et al 1988)
• Sensitivity to DDT GCS and GST (Hung et al.
  2004)
• Bladder Cancer (Hung et al. 2004)
• O.R. GSTM1 1.69
• O.R. GSTT1 1.74
• O.R. GSTM1 Env Exposure 2.77
• Acute leukemia GST

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Breakdown of the defense system

• GSH deficiency
• Genetic (GCS polymorphisms, epigenetic
  dysfunction)
• Environmental (oxidative consumption or
  inflammation)
• GST problems
• Genetic (GST polymorphisms, epigenetic
  dysfunction)
• Environmental (Inflammatory cascade/ARE
  dysfunction)
• Phase III can get blocked and then downregulates
  Phase II enzymes
• Can stop multiple detoxification pathways!

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Biggest Reason for Phase III Dysfunction
Inflammation! Especially in Gut! -Hallmark of
Autism cases -Easily caused by heavy metal
induced oxidative damage
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Coordinated Expression of Phase II and III
MRP2 and GSTp coregulated
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Oxidative Activation
Phase I
Glutathione Conjugation
Sulfation
Glucuronidation
Phase II
Cellular MRP1
Blood
Phase III
OATP
LIVER
MRP2
Normal Small Intestine
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Oxidative Activation
Phase I
Glutathione Conjugation
Sulfation
Glucuronidation
Phase II
Cellular MRP1
Blood
Phase III
OATP
LIVER
MRP2
Inflamed Small Intestine
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Oxidative Activation
Phase I
Glutathione Conjugation
Sulfation
Glucuronidation
Phase II
Cellular MRP1
Negative Feedback Inhibition of Phase II
Blood
Phase III
Inflammation causes Downregulation of MRP2
OATP
LIVER
MRP2
Inflamed Small Intestine
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Oxidative Activation
Oxidative Activation
Oxidative Stress From Phase I/Phase II mismatch
Phase I
Glutathione Conjugation
Sulfation
Glucuronidation
Phase II
Build-up of both cellular and blood-borne toxins
Cellular MRP1
Negative Feedback Inhibition of Phase II
Blood
Phase III
Inflammation causes Downregulation of MRP2
OATP
LIVER
MRP2
Inflamed Small Intestine
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Amalgam as Cause for Inflammation Cysteine Mouth
Rinse
HgII
MeHg
MeHg
MeHg
HgII
HgII
No Amalgam 25ppt MeHg, 5ppt HgII
Amalgam 10ppb
Amalgam 10ppb
Amalgam (2nd Rinse) 16ppb
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MeHg Mitochondrial Inferno Breakdown of the
Thiolic Protection System
Mitochondrial Stress The MeHg-induced Blaze

1. Covering Exposed Thiols
2. Mopping up ROS
3. Binding Hg

Leaking ROS and cyt c
Decay in GSH
Insufficient Protection from GSH
TrX releases ASK1
ROS and Hg Oxidize TrX
Apoptosis Cell Death
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Mercury and The Human Detoxification System

• What the key forms of Hg are
• How the Natural Detoxification System Works
• How the Detox System gets subverted leading to
  biochemical stress
• Mercury Analysis (Blood is not a dirty word)
• Intestinal Metals Detox System to repair and
  amplify the bodies Detoxification System and
  safely remove mercury

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Testing for Hg

1. Ambient Measures
2. Blood
3. Hair
4. Urine
5. Stool
6. Provoked Measure
7. Urine

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Challenge Tests What do they mean? Are They
Necessary?
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Chelation therapy Dump from blood followed by
reloading from cellular burden
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Mercury Speciation Testing

• Separates the two main forms of mercury in the
  human body
• Once separately measured, ambient measurements
  reveal A LOT without challenge tests

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Whole Blood vs. RBC

• Partitioning between RBC and plasma different for
  MeHg and HgII
• MeHg 90 RBC/10 Plasma
• Access to brain, intracellular, metabolic-heme
• HgII 50/50
• Lymph an extension of plasma 5X the volume of
  blood
• Extracellular Matrix, also concept of terrain
• Look at both forms with whole blood

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Inorganic Hg and Poisoning of the Extracellular
MatrixUhhh, What Matrix?
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Blood Testing The Great Tuna Experiment

• 2 cans of Albacore Tuna in one sitting

24 hours later
24 hours later
24 hours later
2 hours later
2 hours later
2 hours later
Pre-Tuna
Initial Decay Redistribution to Tissues
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Walleye Experiment Decay and Body Burden
After initial peak and decay, Blood reflects Body
Burden!
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Mercury Speciation Testing and Compartment Ratio
Testing

• Ambient Measurement Suite
• Blood MeHg HgII
• Hair MeHg only
• -Compare to blood MeHg for excretion measurement
• Urine HgII
• -Compare to blood inorganic mercury for excretion
  measurement

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Dentist Levels - Healthy
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Dentist Levels Chronic Inflammation
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Dentist Levels Kidney Problem
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Patient MeHg Detox Enzyme Defficiency
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Hg Retention HairBlood
Normal Excretion
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Problem of Toxicity Diagnosis

• Levels can not show toxic response
• Toxic response from
• Abilities of your defense systems
• Hyper-sensitivities/Allergies developed during
  exposure
• Must be Diagnosed from combination of
• Clinical Symptomology, and
• Auxiliary Testing

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Auxiliary Testing

• Allergy Testing MELISA, ELISA
• Porphyrins stress to ATP cycle and to kidneys
• GSH Levels antioxidant/detoxifier defense
• GST Activity
• Trx and TrxR
• Genetic Susceptibilities
• GCL, GSTm, GSTp, ApoE, CPox

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Mercury and The Human Detoxification System

• What the key forms of Hg are
• How the Natural Detoxification System Works
• How the Detox System gets subverted leading to
  biochemical stress
• Mercury Analysis (Blood is not a dirty word)
• Intestinal Metals Detox System to repair and
  amplify the bodies Detoxification System and
  safely remove mercury

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Removal of HgAmplifying and Augmenting Natural
Systems
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Biochemical Hg Removal Requirements

• Intracellular Glutathione Sufficiency
• -Liposomal gets in some cells
• -Transpeptidases dissemble/reassemble
• Effective GST Activity (Phase II - mobilization)
• Effective Phase III Clearance including
  intestinal binding

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Product/System for Intestinal Detoxification

• Intestinal Metals Detox (IMD)
• -Phase III opener (dietary supplement)
• Phytonutrients Antioxidant Response Element
• - Upregulate Transcription of Phase II, GSH, SOD
  via ARE
• Source of GSH
• -ReadiSorb Liposomal GSH Livon Labs
• -Nutritional Therapy OSR

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Product/System for Intestinal Detoxification

• Intestinal Metals Detox (IMD)
• Use Intestines NOT Kidneys for Metal Removal
• Insoluble (NON-ABSORBED) silica particles
  saturated with strong (thiol) binding groups
• Binds Mercury in Intestines and moves out of Body
• Interrupts Enterohepatic Circulation
• Opens Phase III transporters
• Bilirubin levels fall dramatically too!
• Then relies on the Natural Detox System (GSH)

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Oxidative Activation
Oxidative Activation
Oxidative Stress From Phase I/Phase II mismatch
Phase I
Glutathione Conjugation
Sulfation
Glucuronidation
Phase II
Build-up of both cellular and blood-borne toxins
Cellular MRP1
Negative Feedback Inhibition of Phase II
Blood
Phase III
Inflammation causes Downregulation of MRP2
OATP
LIVER
MRP2
Inflamed Small Intestine
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Oxidative Activation
Phase I
Glutathione Conjugation
Sulfation
Glucuronidation
Phase II
Cellular MRP1
Blood
Phase III
OATP
LIVER
MRP2
Normal Small Intestine
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Phase III EnhancementBilirubin (unrelated to
GSH) falls too
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A Look at Natural Attenuation Post-Revision
Halbach et al., 2008, Environ Research 10769-78
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Changes in RBC Hg after Dental Revision
Revision-MeHg
No Revision - MeHg
No Revision - HgII
Revision
Revision
Revision
Revision-HgII
Halbach et al., 2008, Environ Research 10769-78
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MeHg Moving from tissues to Bloodstream But NOT
Out!
Modeled Trend -Revision
Modeled Trend - No Revision
Halbach et al., 2008, Environ Research 10769-78
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IMD Decreases Half-Life in Blood
With IMD ½-Life 17 days To Baseline 40 days
No Treatment (Walleye) ½-Life 46-66 days To
Baseline 160 days
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Small Clinical Trial Results
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Patient MeHg Detox Enzyme Deficiency
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Huggins Protocol
Includes Ancestral Diet, Matrix Supplements, IMD,
and OSR
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Product/System for Intestinal Detoxification

• Intestinal Metals Detox (IMD)
• -Phase III opener
• Phytonutrients Antioxidant Response Element
• - Upregulate Transcription of Phase II, GSH, SOD
• Source of GSH
• -ReadiSorb Liposomal GSH
• -Nutritional Therapy

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Phytogenomics

• Certain Phytochemicals upregulate Phase II
  enzymes as well as GSH, SOD
• The Anti-Inflammatory Cascade
• Polyphenols
• Sulfur compounds
• Crucifers
• Garlic oil
• NOT CHELATORS!!!

Also Progesterone and Pregnenolone very strong in
this regard.
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Chemoprevention by Keap1-Nrf2 Signaling pathway
by Phase II Inducers
Kwak et al., 2004, Mutation Research, 555133-148
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Polyphenolics

• Anti-inflammatory cascade
• Upregulate Phase II enzymes through binding to
  membrane and nuclear receptors (transcription
  factors)
• Vascular protective effects (strengthen
  capillaries and improve oxygen delivery)
• Anti-cancer
• Cross BBB

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Flavanols (Polyphenolics)
Ellagic Acid
Epicatechin in tea, cocoa monomer for OPCs
from Pine Bark, Grape seeds,
Quercetin
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Haritaki Terminalia Chebula
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Haritaki Terminalia Chebula

• Aged rates had higher markers of mitochondrial
  free-radical damage
• 2. Also Uptick of CAT and GPx, downturn of
  MnSOD, GR, GST, GSH. Vit C, Vit E
• - normal defenses and export system cant keep
  up wit the load, resulting in more accumulation
  of free-radical generating molecules and more
  reliance on downstream protections
• -further depleting GSH
• 3. ALL MARKERS REVERSED TO YOUNG LEVELS WITH
  HARITAKI

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The Clearing, The Clearing. The Great
Clearing This is the path to fulfillment. So be
it!
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Sulfur Compounds

• Anti-inflammatory cascade
• Upregulate Phase II (and Phase III) enzymes
  through binding to membrane and nuclear receptors
  (transcription factors)
• Vascular protective effects
• Anti-carcinogenic

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Sulfur Compounds
Sulforaphane the famous crucifer compound
Erucin from crucifers not as strong as SF
Allyl-isothiocyanate Oil of mustard,
horseradish
Allicin from garlic
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Product/System for Intestinal Detoxification

• Intestinal Metals Detox (IMD)
• -Phase III opener
• Phytonutrients Antioxidant Response Element
• - Upregulate Transcription of Phase II, GSH, SOD
• Source of GSH
• -ReadiSorb Liposomal GSH or Livon Labs
• -Nutritional Therapy

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Liposomal Glutathione

• Liposomal Encapsulation
• Phospholipid bilayer
• Bypasses peptidases that break down glutahione
• Direct absorption in upper intestine
• Some evidence that they enter cells
• Macrophage oxidative damage protection

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Nutritional GSH Augmentation

• Vitamin C
• Antioxidant Phytonutrients
• Glutathione Precursors
• NAC, glutamate, glycine
• NAC and Vit C
• Whey powder
• GGC gamma glutamyl cysteine (product in
  development from Australia)

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Summary

• Retention Toxicity Related to Dysfunction of
  Natural Glutathione Detox System, BUT Affects
  other detox systems
• Inflammation as major impediment to detox
• MeHg a hidden danger with amalgam
• Hg speciation testing and Compartment Ratio
  Analysis to monitor detox
• Opening channels and upregulating natural system
  a safe and effective detox strategy

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Thank You Huggins Alliance!
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Organic Anion Transporters GSH Ionization in
Aqueous Solutions
H2O
_
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Cruciferous Compounds
Glucosinolate
Sulforaphane the famous crucifer compound
Glucobrassicin
Indole-3-Carbinol
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Transformations of mercury

• Hg0 ? HgII (saliva, blood and tissues)
• HgII ? Hg0 (intestines)
• HgII ? MeHg (intestines)
• MeHg ? HgII (intestines, tissues)
• EtHg ? HgII (tissues, blood)

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Small Clinical Trial Results
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