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

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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