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Determination of Saliva Thiocyanate Concentrations Using Spectrophotometric Analyses and its Correlation to Smoking

Determination of Saliva Thiocyanate Concentrations Using Spectrophotometric Analyses and its Correlation to Smoking. Alberto Juarez Chemistry 03.331 Instrumental Analysis Dr. Salim Diab Fall 2003. Introduction.

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Determination of Saliva Thiocyanate Concentrations Using Spectrophotometric Analyses and its Correlation to Smoking

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  1. Determination of Saliva Thiocyanate Concentrations Using Spectrophotometric Analyses and its Correlation to Smoking Alberto Juarez Chemistry 03.331 Instrumental Analysis Dr. Salim Diab Fall 2003

  2. Introduction • The assessment of the number of juveniles that smoke would be more effective with a quick and efficient chemical test rather than randomized surveys (Luepker et al. 1981). • Smoking has been shown to be a source of cyanide, which results in increased levels of thiocyanate that can be detected spectrophotometrically.

  3. Intro: So what is Thiocyanate anyway? • Thiocyanate (SCN-) is the metabolite of cyanide. • end product of the detoxification of compounds containing cyanide • reaction catalyzed by the enzyme rhodanase • Rhodanase is produced by the mitochondria of living cells

  4. The chemical reaction • Cyanide (CN-) reacts with thiosulfite (S-SO3-2) in a reaction catalyzed by rhodanase to form thiocyanate and sulfite ion (SO3-2) as presented below • CN- + S-SO3-2 SCN- + SO3-2

  5. Do you have Thiocyanate in your system? • Well, yes • Thiocyanate is present in everybody’s saliva • This can only mean that we are exposed to cyanide everyday ;) • One source is FOOD (White et al. 1998) • includes but not limited to, cabbage, broccoli, almond, horseradish, corn, and manioc (cassava).

  6. Another cyanide source is cigarette smoke (Luepkeer RV, et al 1981). • cyanide enters the body mainly through pulmonary absorption but also through the buccal and/or pharyngeal mucosal layers (Galanti 1997)

  7. Purpose • The purpose of this experiment was to find whether or not smokers had statistically higher overall average [SCN-] than non-smokers, thus establishing a correlation between [SCN-] and smoking • Hypothesis • Smokinghigher [CN-]higher [SCN-]

  8. Possible problems • Food containing cyanide may produce false-positive tests. • However it was found that saliva thiocyanate levels in non- and ex-smokers are not affected by alimentary sources (Galanti et al. 1997).

  9. Methods • determination of thiocyanate concentrations through ultraviolet/visible (UV/Vis) radiation spectrophotometry (Lahti et al. 1999) • Method indirectly measures CN- • concentration of thiocyanatoiron ion (FeSCN2+) is determined • It has a deep-red color that absorbs maximally at =447nm.

  10. The Thiocyanatoiron(III) ion Reaction • (remember) • CN- + S-SO3-2 SCN- + SO3-2 • thiocyanate (SCN-) reacts with ferric ions (Fe3+) to form thiocyanatoiron(III) ion (FeSCN2+) • Fe3+ (aq) + SCN- (aq)  FeSCN2+ (aq)

  11. When (Fe3+) is much greater than (SCN-) we can assume that all of (SCN-) is converted to (FeSCN2+) • thiocyanatoiron concentration [FeSCN2+] is equal to the original thiocyanate concentration [SCN-] • A standard curve was prepared to determine the molar extinction coefficient of [FeSCN2+]

  12. Beer’s law, or the Beer-Lambert equation shown below. • A = abc or A = bc

  13. sample size was around 2mL and placed in Eppendorf tubes • centrifuged at 12,000 rpm for 12 min • 200L portion of supernatant was reacted in 5mL of Fe(NO3)3 solution • %T at 447 nm was obtained with a UV/Vis spectrophotometer

  14. Results

  15. non-smoker (N) mean [SCN-] • 2.053 X 103 nmols • smokers (S) mean [SCN-] • 1.465 X 103 nmols • differences in [SCN-] between the two groups was not significant • (P = 0.515).

  16. Discussion and Conclusions • There is no difference in [SCN-] between smokers and non-smokers • May not be suitable test for determining juvenile smoking

  17. The higher [SCN-] in non-smokers suggests there are other variable that affect [SCN-] • Alimentary sources do play a role • SCN- half-life may have an effect

  18. Alimentary Cyanide Sources • Food is still a probable source • Cyanide concentrations in cassava result in health aberrations (White et al. 1998) • Conflicts with Galanti et al. (1997)

  19. Role of SCN- half-life • It may be possible for a smoker to have normal [SCN-], if the analysis is done long enough after the last cigarette was smoked. • Saliva [SCN-] may rise significantly after smoking • after a certain amount of time the saliva [SCN-] is metabolized or excreted

  20. However… • There are various reports on SCN- half-life • The shortest reported is 3 days • Smokers surveyed had smoked that same day • At most 2 hours prior to sampling • SCN- half-life is probably not a factor in this study

  21. Flaws and Improvements • This study worked with a small sample size (n = 7 ) • due in part to people’s apprehension to donate saliva samples (there is something yucky about spiting into a vail : () • the non-smoker group should avoid foods that are known to contain CN- • and the time after smoking a cigarette should be recorded for smokers. • a timed study where [SCN-] is plotted against time after last cigarette

  22. Other implications • spectrophotometric determination of salivary [SCN-] is sensitive • Could potentially be used as a preliminary determinant of cyanide or thiocyanate poisoning in forensic cases • Currently uses blood sampling

  23. Acknowledgments • Dr. Salim Diab • Veronica Cardona • Brian Herbst • Hayden Hollister • Jessica J.

  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. Any Questions?

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