Rate of Metabolism of Cyanide to Thiocyanate in Saliva After Smoking

By:

Matt HerringDeanne Seymour

and

Bettylou Wahl

What is Cyanide? Common forms: HCN, NaCN, KCN Found in foods such as cassava,

lima beans, almonds, and apples Produced by certain bacteria and

fungi Enters the body through ingestion,

inhalation, and absorption

C N- ::

Hydrogen Cyanide Colorless gas Almond scent BP: 25.6 C Enters the body through inhalation Toxic gas present in cigarette smoke Released in metallurgy, electroplating, metal

cleaning processes and car exhaust Used for fumigation of dry foods such as

cereals, seeds, nuts, and tobacco Used for disinfestation of buildings

Effects of HCN on the body Chronic low exposure causes neurological,

respiratory, cardiovascular, and thyroid effects breathing difficulties, heart pains, vomiting, blood

changes, headaches Long term exposure causes central nervous

system effects weakness of digits, difficulty walking, dimness of

vision, deafness High levels of exposure in a short amount of

time harms the brain and heart and may cause coma and death

Cigarette Smoke

Cigarettes are a large source of cyanide

Cyanide is not present in actual cigarettes, but is formed through combustion and found in the smoke

Cyanide levels in inhaled cigarette smoke range from 10 to 400 micrograms per cigarette

Thiocyanate Cyanide is metabolized to less toxic

thiocyanate through sulfuration with thiosulfate by mitochondrial rhodanase in the liver

CN- + S2O3-2 SCN- + SO3

-2

Thiocyanate is present normally in human saliva at approximately [0.01%]

Thiocyanate levels in saliva have been found correlate with cyanide intake

Methods for determining cyanide and thiocyanate levels HS-GC (head-space gas chromatography) Spectrophotometric Konig method

Thiocyanate ion (SCN-) reacts with iron Fe3+ to yield FeSCN2+ complex which can be detected spectrophotometrically (at 448 nm)

The complex exhibits a red/orange color that becomes darker with greater concentration

UV-VIS (HP) Instrument is used to measure the absorption of FeSCN2+ complex, which correlates to the [SCN-]

Previous research and studies Saliva thiocyanate levels of

smokers, non-smokers, and second hand smokers have been studied

Smokers have been found to have higher overall levels of thiocyanate than non-smokers (Lahti et. al. 1999)

Challenges Establishing an accurate calibration

curve Monitoring peoples’ diets for testing Storing the samples Obtaining a large enough sample

size Finding a strong control when there

are many variables

Our Research Initially, saliva samples from smokers

and non-smokers were analyzed (Juarez 2004)

In order to confirm past research, we set out to see if there were any significant differences in thiocyanate levels between smokers and non-smokers

Method for preparing saliva Obtain 2.5 mL of saliva Centrifuge at 12,000 rpm for 12 min Remove and centrifuge clear liquid

again at 12,000 rpm for 12 min Add 0.5 mL of centrifuged saliva to

9.5 mL of 0.0019 M Fe(NO3)3 Measure absorption at 448 nm in

spectrophotometer

Instrumental Detection Levels

HP 8452A Diode Array Spectrophotometer

Absorbance 0.574 0.246 9.38E-02 1.10E-02 -3.00E-02

Concentration (M) 2.00E-05 1.00E-05 5.00E-06 2.50E-06 1.25E-06

Establishing Standard Curve Beer’s Law: A=abc Used to determine the concentration

from the experimental absorption level values

Established using five known concentrations of FeSCN as standards

Curve checked for accuracy

Standard Calibration Curve

0.00

0.02

0.04

0.06

0.08

2.00E-05 4.00E-05 6.00E-05 8.00E-05 1.00E-04

Concentration of FeSCN+2(M)

Abs

orba

nce

Results

SCN- concentration (M)

SmokersNon-Smokers

4.57E-03 1.65E-02

2.65E-03 1.42E-02

9.41E-03 5.34E-03

6.77E-03 1.08E-02

5.81E-03

7.73E-03

8.90E-03

2.62E-03

Average SCN Concentration of Smokers and Non-Smokers

0.00E+00

2.00E-03

4.00E-03

6.00E-03

8.00E-03

1.00E-02

Smokers Non-smokers

SC

N C

on

cen

trat

ion

(M

)

Troubleshooting Results did not show higher overall

thiocyanate levels for smokers Not enough samples analyzed Problem with our methods? Initially samples were take right

after the subject smokes Did this allow ample time for

cyanide to be metabolized after smoking?

New Scope of Investigation Set to find any change in

thiocyanate levels over time after the subject smokes

If any changes are observed, then the variable of time must be taken into account

Round 2: Rate of Metabolism Saliva samples were taken before

smoking and after smoking at set time intervals (initially, 30 minutes, 60 minutes)

Changes in thiocyanate concentrations over time will allow us observe both the rate of metabolism and degradation of thiocyanate in the saliva

More ResultsChange in SCN Concentration Before

and After Smoking

0.00E+00

1.00E-03

2.00E-03

3.00E-03

4.00E-03

5.00E-03

6.00E-03

BeforeSmoking

Initial 30Minutes

60Minutes

SC

N c

on

cen

trat

ion

(M

)

Smokers

-0.002

0

0.002

0.004

0.006

0.008

0.01

B4 S 0 Min 15 Min 30 Min 45 Min 60 Min

Time

Con

cent

ratio

n of

Fe

SC

N+2

Bettylou

Sub. #1

Sub. #2

Bettylou

Bettylou

Sub. #3

Sub #10

Sub#12

Summer ’05

Non-smokers

-0.001

0

0.001

0.002

0.003

0.004

0.005

0.006

B4 S 0 Min 15 Min 30 Min 45 Min 60 Min

Time

Con

cent

ratio

n of

Fe

SC

N+2

Matt

Matt

Sub #4

Sub #5

Sub #6

Sub #7

Sub #8

Sub #9

Sub#11

Sub#13

Sub#14

Summer ‘05

Future Goals Reconfirm the rate study of

metabolism of cyanide to thiocyanate

Once again take up our previous research involving the comparisons of smokers and non-smokers with a greater degree of accuracy

Other Future Projects Analyze the amount of cyanide

intake from certain foods and vitamins (B12) compared to tobacco smoke

Amount of cyanide in cigarette smoke compared to things such as vehicle exhaust, metal industry emissions, etc.

Acknowledgements USF for the use of its

instrumentation Dr. Frank Pascoe, Dean of Arts and

Sciences for his grant support Alberto Juarez, USF graduate, for

his work on phase I of this project Dr. Salim M. Diab, Team supervisor

References Galanti LM. Specificity of salivary thiocyanate as marker of cigarette smoking is not

affected by alimentary sources. Clin. Chem., 1997 Jan; 43(1):184-5. Lahti M, Vilpo J, Hovinen J. Spectrophotometric determination of thiocyanate in

human saliva. J Chem Ed. 1999 Sept;76(9): 1281-3 Luepker RV, Pechacek TF, Murray DM, Johnson CA, Hund F, Jacobs DR. Saliva Thiocyanate: a chemical indicator of cigarette smoking in adolescents. Am J Public Health. 1981 Dec;71(12):1320-4. O S Oluwole, A O Onabolu, I A Cotgreave, H Rosling, A Persson, and H Link Incidence of endemic ataxic polyneuropathy and its relation to exposure to cyanide

in a Nigerian communityJ. Neurol. Neurosurg. Psychiatry, Oct 2003; 74: 1417 - 1422.

White WLB, Arias-Garzon DI, McMahon JM, and Richard T. Sayre Cyanogenesis in Cassava: The Role of Hydroxynitrile Lyase in Root Cyanide

ProductionPlant Physiology, Apr 1998; 116: 1219 - 1225.

Wood John L. and Edward F. Williams, Jr. THE METABOLISM OF THIOCYANATE IN THE RAT AND ITS INHIBITION BY

PROPYLTHIOURACILJ. Biol. Chem., Jan 1949; 177: 59 - 67.

http://www.acsu.buffalo.edu/~koudelka/kinetics/kineticsproblemset1answers.pdf http://www.rxlist.com/cgi/generic3/nitroprusside_cp.htm

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