Detecting Human Exposure to Environmental Toxins · 4/3/2014 · Case 3: Rosary Pea 2013, Texas Rosary Pea necklace 22-month-old ingested ~20 peas Patient began vomiting 6 hours

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Detecting Human Exposure to Toxins

Lucas Zarwell DC Office of the Chief

Medical Examiner

10

Elizabeth Hamelin Centers for Disease

Control and Prevention

4/3/2014

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Acute Exposure

Symptoms

dose effect

? Chronic Exposure

Case Study: Symptomology

Case 1

Severe gastrointestinal illness

Case 3

Severe vomiting

Case 4

Large blisters on arm

Case 2

Dizziness Tingling of lips

Floating sensation

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Ob

serv

ati

on

s

Diagnosis

Visual cues Questions & answers

General marker Impacted by many sources

Unique marker Only a few sources

Why do we need a specific test?

Identify toxin

Evaluate long-term health effects

Determine exposed vs. unexposed

Determine geographical distribution

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Case Study: Toxins

Ricin

Abrin

Saxitoxin

Sulfur Mustard, toxicant

Absorption Ingestion Inhalation Injection

Elimination Distribution

Excretion Detoxification

Reabsorption

Toxication Distribution

Absorption

16

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Toxicant Excretion

In vitro

In vivo

Human exposure

Time (hours) 0 50 100 150 200

Co

ncen

trati

on

(arb

itra

ry)

0

2

4

6

8

10 Toxic Compound or Marker

Metabolite

Adduct

Specific enzyme

Animal studies

Exposure events

What? * Where? * How much? * How long?

Method Application

2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8

Time, min

0.0

6.0e5

1.2e6

1.8e6

2.4e6

3.0e6

Inte

nsity,

cp

s

Symptomatic

Asymptomatic

Identify compound in poisoning scenarios

Differentiate between exposed/unexposed

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Analytical method development

IS

Matrix

MS HPLC

SPE Evaporation Reconstitution

Sample preparation Separation Detection

Online SPE

IS

Matrix

IS

Matrix

Dilute and shoot

Challenges: Potential False Positives

Similar compounds 2-CHA metabolite from riot control agent CS 4-CHA metabolite from pharmaceutical

Analyte

Matrix

Matrix components

Urinary component at same transition

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Challenges: Potential False Negatives

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

Inte

nsity

, cp

s

In Matrix In Solvent

0%

20%

40%

60%

80%

100%

120%

Water Urine Serum

Strata X

WAX

SAX

ZrO2

Si

Ave

rag

e E

xtra

ctio

n E

ffic

ien

cie

s

Extraction efficiency

Matrix effects

Audience Trivia Question

What is the source of the toxin ricin?

Tree Bark

Rosary Peas

Castor Beans

Bamboo Rice

Silver Beatles

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Case 1: Ricin Suicide Attempt 2005, California

58-year-old homeless man, chewed 6 castor seeds

LD50

Inhalation or injection:

5-10 mg/kg

Higher for ingestion

Ricin Ricinine

Ricinine method

QL QH

Precision 14% 0.78%

Accuracy 106% 102%

Lowest Reportable 0.03 ng/mL

% Ricin

% Ricinine -

% RCA 5% of castor bean

20% of ricin content 1% of castor bean

Ricin toxicity = 5.0 mg/kg

Method Performance

+

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Time after ingestion (hrs)

0 20 40 60 80

ug

Ric

inin

e/g

Cre

ati

nin

e

100

200

300

400

500

600

700

800

~ 6 Castor Seeds

Castor Bean Ingestion Analysis of Ricin Biomarker in urine

Ricinine: Other Sources

Present in cosmetics, oils, and pharmaceuticals

12 positive of 989 urine samples (1.2%)

Must take analytical results in context with symptomology

0

2

4

6

1 3 5 7 9 11

Results ranged from 0.186-4.15 ng/mL

Ric

inin

e (n

g/m

L)

Sample

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Case 2: Saxitoxin poisoning from shellfish 2011, Alaska

Happy Clams Red Tide w/Toxins Clams w/Toxins

Captured Clams Happy People

Consume Clams Unhappy People

w/Toxins

Two persons present with dizziness, shortness of breath, nausea, floating sensation following ingestion of mussels, which they had

harvested in the wild

Artwork by Ken Hsuan Liu

Saxitoxin in Urine

XIC of +MRM (5 pairs): 300.100/282.100 Da from Sample 13 (Curve 6_RR_13) of Curve 6.wiff (Turbo Spray) Max. 4.3e5 cps.

2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

In

te

ns

ity

, c

ps

3.26

Saxitoxin

Internal Standard

QL QH

Precision 8.0% 5.5%

Accuracy 99.3% 99.6%


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Audience Trivia Question

About how much water is in urine?

73%

80%

84%

92%

95%

Southeast Alaska Outbreak May – June 2011

MMWR…http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6045a3.htm#tab.

Patient Symptoms

Time from consumption to symptom

onset

Type of shellfish consumed

Toxin level in shellfish consumed (µg

saxitoxins/100 g of meat*)

Toxin level in urine (ng/mL)

Hospitalized

A

Ataxia, difficulty swallowing, dizziness, difficulty moving, floating sensation,

nausea, paresthesia, shortness of breath, weaknes

3.5--4 hrs Cockles 528 N/A Yes (ICU)

B Paresthesia 10--15 min Cockles 528 N/A No

C Paresthesia 2.5--3 hrs Cockles 528 N/A No

D Ataxia, dysphagia, floating sensation,

paresthesia, weakness <1 min Cockles 528 N/A No

E Paresthesia <1 min Cockles 528 N/A No

F Ataxia, dysphagia, floating sensation,

paresthesia, shortness of breath, weakness

<1 min Blue mussels 5,037 118 Yes (ICU)

G Dizziness, dysphagia, floating sensation, nausea, weakness

1 hr Blue mussels 5,037 15 Yes

H Ataxia, dizziness, floating sensation,

paresthesia, vomiting, weakness 45 min

Butter/ Little neck clams

1,321 N/A Yes

Abbreviations: N/A = not available; ICU = Intensive-care unit. * The international regulatory action level set by the U.S. Food and Drug Administration for paralytic shellfish toxins in shellfish is ≥80 µg

saxitoxins/100 g of meat

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Urine samples analyzed at CDC 2 positive for saxitoxin

at 118 and 15 ng/mL

2 negative

All shared the same meal

Ketchikan Mussels

Southeast Alaska Outbreak May – June 2011

Case 3: Rosary Pea 2013, Texas

Rosary Pea necklace

22-month-old ingested ~20 peas

Patient began vomiting 6 hours after ingestion

with an LD50 of 0.7 µg/kg of body mass when given to mice intravenously,[1] and 10–1000 µg/kg when ingested by humans and 3.3μg/kg when inhaled by humans

Toxin: Abrin

Found in Abrus precatorius

LD50

0.04 mg/kg

http://en.wikipedia.org/wiki/LD50

http://en.wikipedia.org/wiki/LD50

4/3/2014

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Abrine method

% Abrin -

% Abrine -

0.12% of bean

~2x abrin 0.45% of bean

Method Performance

Abrine

QL QH

Precision 4.6% 7.5%

Accuracy 100% 101%

Lowest Reportable 5 ng/mL

N

C

NCH3

O

OH

L-Abrine

Urinary Excretion

Results from Exposure

Exposure sample: 7,703 mg/g creatinine

Background levels: 0.44 mg/g creatinine (n=113)

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Case 4: Old munitions 2004, Delaware

Biomarkers of Exposure

SOHHO

S

O O

SH3C S CH3

1, 1-sulfonylbis[2-(methylthio)ethane] (SBMTE)

Thiodiglycol (TDG) HD

Urinary Excretion

Background levels 0.5-20 ng/mL

Protein Adduction Albumin

Sulfur Mustard

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0

60

110

160

210

0 1 2 3 4 5 6 7 8 9 10

Uri

na

ry S

BM

TE

(n

g/L

)

Days following exposure

Measurement of Exposure Sulfur Mustard

SBMTE

QL QH

Precision 9.5% 7.8%

Accuracy 105% 101%


Lessons Learned

Choose biomarker well Minimal sources

Maximum time

Specific tests should be well characterized Characterize matrix

Minimize false positives

Minimize false negatives

Determine sensitivity

Evaluate method thoroughly

4/3/2014

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Acknowledgements

Chariety Sapp

Rudolph Johnson

Jerry Thomas

Chris Pittman

Joe Wooten

Will Bragg

Brian Crow

Doris Ash

Thank you!

For more information please contact Centers for Disease Control and Prevention

1600 Clifton Road NE, Atlanta, GA 30333

Telephone: 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348

E-mail: [email protected] Web: http://www.cdc.gov

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

National Center for Environmental Health

Division of Laboratory Sciences

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Medical Examiner

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Detecting Human Exposure to Environmental Toxins · 4/3/2014 · Case 3: Rosary Pea 2013, Texas Rosary Pea necklace 22-month-old ingested ~20 peas Patient began vomiting 6 hours