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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?. 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

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  1. Rate of Metabolism of Cyanide to Thiocyanate in Saliva After Smoking By: Matt Herring Deanne Seymour and Bettylou Wahl

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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-]

  8. 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)

  9. 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

  10. 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

  11. 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

  12. Instrumental Detection Levels HP 8452A Diode Array Spectrophotometer

  13. 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

  14. Results SCN- concentration (M)

  15. 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?

  16. 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

  17. 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

  18. More Results

  19. 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

  20. 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.

  21. 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

  22. 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

  23. The end

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