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Rate of Metabolism of Cyanide to Thiocyanate in Saliva After Smoking. By: Matt Herring Deanne 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

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rate of metabolism of cyanide to thiocyanate in saliva after smoking

Rate of Metabolism of Cyanide to Thiocyanate in Saliva After Smoking

By:

Matt Herring

Deanne Seymour

and

Bettylou Wahl

what is cyanide
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
hydrogen cyanide
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
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
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
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
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
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
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
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
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
Instrumental Detection Levels

HP 8452A Diode Array Spectrophotometer

establishing standard curve
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
results
Results

SCN- concentration (M)

troubleshooting
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
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
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
future goals
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
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
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
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