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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. Summer ’05

  20. Summer ‘05

  21. 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

  22. 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.

  23. 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

  24. 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

  25. The end

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