Spectrophotometric Analysis of Thiocyanate in Saliva and its Correlation to Tobacco Smoking

1. Spectrophotometric Analysis of Thiocyanate in Saliva and its Correlation to Tobacco 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. 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 • However, our results did not confirm previous literature that smokers have a higher thiocyanate concentration than non-smokers

10. Round 2 • Saliva samples were taken before and after smoking at 15 minute time intervals from 0-60 minutes • Changes in thiocyanate concentrations over time will allow us observe both the rate of metabolism and degradation of thiocyanate in the saliva

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 460 nm in spectrophotometer

12. Instrumental Detection Levels HP 8452A Diode Array Spectrophotometer

14. Mean Thiocyanate Concentration of Smokers and Non-Smokers Over Time

15. Mean Thiocyanate Concentration for Each Subject Smokers Non-Smokers

16. Conclusions • No significant difference in saliva thiocyanate concentration between smokers and nonsmokers at each sample time and as a mean for each subject over all sample times • Saliva thiocyanate levels vary more person to person than within each subject over time • No set saliva thiocyanate concentration level to differentiate between a smoker and non-smoker

17. Acknowledgements • USF for the use of its instrumentation • Dr. Frank Pascoe, Dean of Arts and Sciences for his grant support • Dr. Salim M. Diab, Team supervisor • Volunteers, for without them, our research would not exist

18. 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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