o n t e n t - vitamin c foundation

Looking Inside the Black Box: Understanding Analytical Approaches,

Diagnostic Tools, and Detoxification Strategies for Mercury Intoxication

Christopher W. Shade, [email protected]

Quicksilver Scientific, L L CL afayette, CO 80026

(303)263-6903Heretical C

ontent

Mercury and The Human Detoxification System

1. What the key forms of Hg are 2. How the Natural Detoxification System

Works3. How the Detox System gets subverted

leading to biochemical stress4. Mercury Analysis (Blood is not a dirty word)5. Intestinal Metals Detox System to repair

and amplify the bodies Detoxification System and safely remove mercury

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

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

Targets of mercury forms

• Brain/CNS• Kidney• Liver• Heart• Pituitary/Thyroid

– And thus all glandular

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)

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

Inorganic Hg and Poisoning of the Extracellular Matrix

Defense – Glutathione System Antioxidant, Detoxification, Protein Repair

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

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?

• Detoxification Phases I, II, III– Phase I is an activation, – Phase II is conjugation– Phase III is transport (recently

delineated; control point)

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


• 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)


• 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


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)

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

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!

Biggest Reason for Phase III Dysfunction

Inflammation!

Especially in Gut!-Hallmark of Autism cases

-Easily caused by heavy metal induced oxidative damage

Coordinated Expression of Phase II and III

MRP2 and GSTπ coregulated

Phase I

Phase III

Phase IIGlutathione Conjugation

Sulfation Glucuronidation

OATPB

lood

LIVER

MRP2

Normal Small Intestine

Cellular MRP1

Oxidative Activation

Phase I

Phase III



OATPB

lood

LIVER

MRP2

Inflamed Small Intestine

Cellular MRP1


Phase I

Phase III



OATPB

lood

LIVER


Cellular MRP1


Inflammation causes

Downregulation of MRP2

Negative Feedback – Inhibition of Phase II

MRP2

Phase I

Phase III

Phase II


Glutathione Conjugation


OATPB

lood

LIVERMRP2


Cellular MRP1


Inflammation causes


Oxidative Stress From Phase I/Phase II mismatch

Build-up of both cellular and blood-borne toxins


Amalgam as Cause for Inflammation Cysteine Mouth Rinse

[min.]Time

620 640 660 680 700

[V]

Volta

ge0.0

0.5

1.0

C:\Clarity\WORK1\20090224_Chrom_200908_0023 ARDL Waters

614.

810

48

627.

340

49

630.

537

50

644.

467

51

648.

993

52

663.

460

53

667.

730

54

680.

447

55

685.

300

56

699.

743

57

702.

963

58

[min.]Time

1540 1560 1580 1600 1620

[V]

Volta

ge

-0.5

0.0

0.5

1.0


1539

.837

1

05

1553

.067

1

06

1557

.593

1

07

1570

.753

1

08

1575

.140

1

09

1589

.303

1

10

No Amalgam 25ppt MeHg, 5ppt HgII

Amalgam – 10ppbAmalgam (2nd Rinse) – 16ppb

[min.]Time

620 640 660 680 700

[V]

Volta

ge0.0

0.5

1.0


614.

810

48

627.

340

49

630.

537

50

644.

467

51

648.

993

52

663.

460

53

667.

730

54

680.

447

55

685.

300

56

699.

743

57

702.

963

58

Amalgam – 10ppb

MeHg

HgII

MeHg

HgII

MeHg

HgII

MeHg Mitochondrial Inferno – Breakdown of the Thiolic Protection

SystemMitochondrial Stress –

The MeHg-induced Blaze

Leaking ROS and cyt c

ROS and Hg Oxidize TrX

1) Covering Exposed Thiols

2) Mopping up ROS

3) Binding Hg

TrX releases ASK1

Decay in GSH

Insufficient Protection

from GSH

Apoptosis – Cell Death

Testing for Hg1. Ambient Measures

1. Blood2. Hair3. Urine 4. Stool

2. Provoked Measure1. Urine

Testing for Hg

1. Ambient1. Blood – MeHg + HgII (MeHg larger)

2. Hair – MeHg only3. Urine – HgII (little bit of MeHg)4. Stool – MeHg + HgII

2. Provoked1. Urine – MeHg + HgII

H

HH

Hg

Challenge Tests – What do they mean? Are They Necessary?

Mercury Industry Workers

Dentists

Controls (Amalgam)

Amalgam-free Referents

Chelation therapy – Dump from blood followed by reloading from cellular

burden

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

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

Inorganic Hg and Poisoning of the Extracellular Matrix

…Uhhh, What Matrix?

MethylMercuryThe Unsuspected Factor H

HH

Hg

1. Direct toxicity not as high as Hg(II)

1. One binding site vs. 2 major and 2 minor

2. BUT…has many insidious properties

3. Is slow to excrete bank of mercury

1. Constant source of inorganic mercury from breakdown

1. Can continue symptoms of hypersensitivity from amalgam after amalgams removed

4. Has “all-access pass”

1. Intracellular, BBB, placental barrier

5. Can form allergy that will persist till all gone

6. If phase II enzymes weak, can stay for long, long time

Accumulation

Enterohepatic Circulation of MeHg

H

HH

Hg

Blood Testing

• Old Dictum – blood is only recent exposure, 3-days• Reality

– 3-day residence only the quick decay after a large dose

– For MeHg, Steady state develops after initial decay; Then blood reflects body burden!

• Real Problem– Most labs detection limits too high to see

dynamics– Need sensitive equipment!

H

HH

Hg

Blood Testing: The Great Tuna Experiment

• 2 cans of Albacore Tuna in one sitting

2 hours later

24 hours later

2 hours later

24 hours later

0.00

0.50

1.00

1.50

2.00

2.50

10/26/2008 10/27/2008 10/28/2008 10/29/2008 10/30/2008 10/31/2008

Bloo

d M

eHg

(ng/

mL)

2 hours later

24 hours later

Initial Decay – Redistribution to Tissues

Pre-Tuna

The Great(er) Walleye Experiment

*Nothing is new under the sun*Clarkson et al., Arch. Env. Health, 1980

Walleye Experiment – Decay and Body Burden

After initial peak and decay, Blood reflects Body Burden!

Mercury Speciation Testing

H

HH

Hg

[min.]Time

4020 4030 4040 4050 4060

[mV]Vo

ltage

-600

-400

-200

0

200 My GC - 6 (05.August 2008)

4019

.867

3

41

4024

.440

3

42

4037

.647

3

43

4042

.010

3

44

4055

.427

3

45

4059

.930

3

46

MeHg

HgII

MeHg

HgII

H

HH

Hg

Testing for MeHg

Mercury Speciation Testing and Compartment Ratio Testing

Ambient Measurement Suite1. Blood – MeHg + HgII

2. Hair – MeHg only-Compare to blood MeHg for excretion

measurement

1. Urine – HgII

-Compare to blood inorganic mercury for excretion measurement

Dentist Levels - Healthy

Dentist Levels – Chronic Inflammation

Indication of Mercury Excretion Ability

0

4 00

-1 2 0 -1 00 -8 0 -6 0 -4 0 -2 0 0 2 0

Blood Me Hg (ng /mL)

Below Average Excretion of blood mercury through the hair.

Average Excretionof blood mercury though the hair (290:1).


0

400

800

1200

1600

2000

2400

2800

0 2 4 6 8 10

Blood MeHg (ng/m L)

Hai

r MeH

g (n

g/g)

Dentist Levels – Kidney Problem

0 0.5 1 1.5 2 2.5

Concentration of Mercury (ng/m L or ug/L)

HgT

Hg(II)

MeHg

Blood Mercury

Patient BloodQS Average


0

4 00

-1 2 0 -1 00 -8 0 -6 0 -4 0 -2 0 0 2 0

Blood Me Hg (ng /mL)

Average Excretionof blood mercury though the hair (290:1).

Below Average Excretion of blood mercury through the hair.


0

400

800

1200

0 1 2 3 4 5

Blood MeHg (ng/m L)

Hai

r MeH

g (n

g/g)

Indication of Kidney Mercury Excretion Ability

0.000.501.001.50

2.002.503.003.504.004.505.00

0 0.5 1 1.5

Blood Hg(II) (ng/m L)

Urin

e H

g(II)

(n

g/m

L)

Good Hg(II) Excretion Line (7:1)

Patient – MeHg: Detox Enzyme Defficiency

Hg Retention – Hair:Blood

0

200

400

600

800

1000

1200

0.000 1.000 2.000 3.000 4.000 5.000

Blood MeHg (ng/m L)

Hai

r MeH

g (n

g/g)

0

100

200

300

400

500

600

700

0.000 1.000 2.000 3.000 4.000 5.000

Blood MeHg (ng/mL)

Hai

r:Blo

od M

eHg

ratio

Normal Excretion

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

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

Removal of HgAmplifying and Augmenting

Natural Systems

Biochemical Hg Removal Requirements

1. Intracellular Glutathione Sufficiency-Liposomal gets in some cells-Transpeptidases dissemble/reassemble

1.Effective GST Activity (Phase II - mobilization)

2.Effective Phase III Clearance including intestinal binding

Product/System for Intestinal Detoxification

1. Intestinal Metals Detox (IMD)-Phase III opener (dietary supplement)

1. Phytonutrients – Antioxidant Response Element

- Upregulate Transcription of Phase II, GSH, SOD via ARE

1. Source of GSH-ReadiSorb Liposomal GSH; Livon Labs-Nutritional Therapy; OSR


• 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)

Phase I

Phase III

Phase II


Glutathione Conjugation


OATPB

lood

LIVERMRP2


Cellular MRP1


Inflammation causes


Oxidative Stress From Phase I/Phase II mismatch

Build-up of both cellular and blood-borne toxins


Phase I

Phase III



OATPB

lood

LIVER

MRP2


Cellular MRP1


Phase III EnhancementBilirubin (unrelated to GSH) falls too

Table 2. Bilirubin Levels on Select Patients with Elevated Blood Bilirubin (From Clinics 1 & 2, 7-10 interventions)

Before After % Change 2 1.1 -45

1.7 0.9 -47.1 1.4 1.3 -7.1 1.2 0.9 -25.0 3.4 2.6 -23.5 1.7 1.2 -29.4

Phase I

Phase III



OATP

Bloo

d

LIVER

MRP2


Cellular MRP1


Halbach et al., 2008, Environ Research 107:69-78

A Look at Natural Attenuation Post-Revision


Changes in RBC Hg after Dental Revision

No Revision - MeHg

Revision

Revision-MeHg

RevisionRevisionRevision-HgII

No Revision - HgII

Modeled Trend -Revision

Modeled Trend - No Revision


MeHg Moving from tissues to Bloodstream… But NOT Out!

Blood Stream Decrease with Quicksilver IMD

0

0.2

0.4

0.6

0.8

1

1.2

1.4

11/24/2007 1/13/2008 3/3/2008 4/22/2008 6/11/2008 7/31/2008 9/19/2008 11/8/2008

Blo

od M

eHg

(ng/

mL)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Blo

od H

gII (

ng/m

L)

MeHg HgII

Depressed Phase II Transferases keep MeHg in cells; Once they kick in again, enterohepatic circulation retards excretion from body

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

0

0.2

0.4

0.6

0.8

1

1.2

1.4

08/10/08 08/20/08 08/30/08 09/09/08 09/19/08 09/29/08 10/09/08 10/19/08 10/29/08

Blo

od M

eHg

(ng/

mL)

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

Feca

l MeH

g (n

g/g-

ww

)

blood MeHgFecal MeHg Excretion

Small Clinical Trial Results

Table 3.1 Changes in blood mercury levels during 7-10 day intervention with amalgam revision and nutritional support.

Clinic 1 Clinic 2 IMD (n=8) No Treatment (n=4) IMD only (n=5) IMD+OSR (n=7) %Decr HgT 23.9 0.4 16.3 17.5 %Decr MeHg 22.9 5.1 15.9 32.3 %Decr HgII 12.4 -4.1 7.1 -15.0

Individual Trial Results

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5


HgT

Hg(II)

MeHg

Before Blood Mercury Comparison

3/14/200912/10/2008QS Average

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7


HgT

Hg(II)

MeHg


8/8/20081/23/2009QS Average

7 months while continuing to eat fish

3 months, stopped fish consumption

Patient – MeHg: Detox Enzyme Deficiency

Patient – MeHg: Detox Enzyme Deficiency

Amalgam Removal and 5 months IMD w/ phytonutrients and nutritional therapy

0 2 4 6 8 10 12 14 16 18 20

1

2 2/12/209

4/16/2009

5/7/2009

7/8/2009

8/25/2009

Huggins Protocol

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6

Concentration of M ercury (ng/m L or ug/L)

HgT

Hg(II)

MeHg

Blood Mercury Comparison

2/23/20097/6/2009QS Average

Includes Ancestral Diet, Matrix Supplements, IMD, and OSR


1. Intestinal Metals Detox (IMD)-Phase III opener


- Upregulate Transcription of Phase II, GSH, SOD

1. Source of GSH-ReadiSorb Liposomal GSH-Nutritional Therapy

“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.

Chemoprevention by Keap1-Nrf2 Signaling pathway by Phase II Inducers

Kwak et al., 2004, Mutation Research, 555:133-148

Putting it All TogetherMitochondrial Stress – The Hg-induced Blaze

Leaking ROS and cyt c

ROS and Hg Oxidize TrX

1) Covering Exposed Thiols

2) Mopping up ROS

3) Binding Hg

TrX releases ASK1

Decay in GSH

Insufficient Protection

from GSH

Apoptosis – Cell Death

Decrease Exposure/ Increase Export

Increase GSH Enzyme Activity

Increase Trx and TrxR

Increase GSH synthesis and Recycling

Put out the Fires – Save the cells

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

Flavanols (Polyphenolics)

Epicatechin – in tea, cocoa; monomer for OPC’s from Pine Bark, Grape seeds,

Ellagic Acid

Quercetin

Haritaki –

Terminalia Chebula

1. 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 can’t 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

Haritaki –

Terminalia Chebula

The Clear ing, The Clear ing. The Great Clear ing

This is the path to fulfillment.

So be it!

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

Sulfur CompoundsSulforaphane – the famous

crucifer compound

Allicin – from garlic

Allyl-isothiocyanate – “Oil of mustard”, horseradish

Erucin – from crucifers; not as strong as SF


1. Intestinal Metals Detox (IMD)-Phase III opener


- Upregulate Transcription of Phase II, GSH, SOD

1. Source of GSH-ReadiSorb Liposomal GSH or Livon Labs-Nutritional Therapy

Liposomal Glutathione

Unilamellar liposomes

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

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)

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

Thank You Huggins Alliance!

Organic Anion Transporters: GSH Ionization in Aqueous Solutions

_

H2O

Mercuric DiGlutathione DiAnionHg(GSH)2

-2 - Soluble, mobile, recognizable

Hg

-2

MethylMercuric Glutathione MonoAnion

CH3Hg(GSH)-1 - Soluble, mobile, recognizable

-1

HgCH3

Cruciferous Compounds

Sulforaphane – the famous crucifer compound

Glucosinolate

GlucobrassicinIndole-3-Carbinol

Transformations of mercury

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

Small Clinical Trial Results

Table 3.1 Changes in blood mercury levels during 7-10 day intervention with amalgam revision and nutritional support.

Clinic 1 Clinic 2 IMD (n=8) No Treatment (n=4) IMD only (n=5) IMD+OSR (n=7) %Decr HgT 23.9 0.4 16.3 17.5 %Decr MeHg 22.9 5.1 15.9 32.3 %Decr HgII 12.4 -4.1 7.1 -15.0

Individual Trial Results

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5


HgT

Hg(II)

MeHg


3/14/200912/10/2008QS Average

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7


HgT

Hg(II)

MeHg


8/8/20081/23/2009QS Average

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