A Comparative Study of Toxic Thiocynate Ion in the Saliva of Smokers and Non-Smokers

Lynn Krebs

ABSTRACT

            For years there have been warnings against smoking and its detriment to one�s health.  I pose the question, is it just as dangerous to be the one who is exposed to the secondhand smoke as it is to be the tobacco user?  To answer this question, the relative amounts of toxic thiocyanate ion in saliva obtained from a variety of individuals are compared.  In order to determine these amounts, absorption spectrophotometry is used in the analysis of the FeSCN²⁺ complex.  This question is of importance because it offers support for the argument that smoking is hazardous to everyone�s health.

 INTRODUCTION

             This study intends to analyze by spectrophotometric methods, the amount of thiocyanate ion present in eleven subjects with differing exposures to tobacco smoke.  It was reported that high concentrations of HCN, a toxic gas, are present in cigarette smoke (Surgeon General, 1990, 35).  The ingestion of this smoke leads to the formation of the thiocyanate ion  (SCN^-) in the liver.  Since the HCN is present in the cigarette smoke and not directly in the cigarette, this can be a potential hazard to the nonsmoker as well, in the form of secondhand smoke.  Secondhand smoke is �a dilute mixture of mainstream smoke exhaled by smokers and sidestream smoke from the burning end of a cigarette or other tobacco product and it is chemically similar to the smoke inhaled by smokers� (www.tobacco.org 4/11/01).  This secondhand smoke accounts for approximately 3,000 lung cancer deaths annually and 37,000 heart disease death in non-smokers (American Lung Association Fact).

            Tobacco smoke contains HCN gas.  Once this gas is ingested it is dissociated into its components according to the following equilibrium equation:

                                  HCN(aq) �� H⁺(aq) + CN^-(aq)                 Equation 1

 The CN^-  ion is then converted into the thiocyanate ion when it reaches the liver.  The thiocyanate ion reacts with the Fe³⁺ ion to yield the FeSCN²⁺ complex that can be detected spectrophotometrically.

            In order to detect the amount of thiocyanate present in the saliva, the clear solution collected from centrifuging the saliva and a 0.0038 M solution of Fe(NO₃)₃ in 1M aqueous HNO₃ are mixed together.  When combined, the iron forms a complex with the thiocyanate ion as stated below.

                            Fe³⁺(aq) + SCN^-(aq) � FeSCN²⁺(aq)                  Equation 2

This complex exhibits a red-orange color.  The deeper the color, the more concentrated the solution is.  This color difference allows for the analysis by visible spectroscopy.

            The UV-VIS Spectrophotometer is used to measure the absorbances of colored solutions at a variety of wavelengths.  The darker the color is, the higher the absorbance value is.  In this study, the absorbances of several solutions consisting of varying concentrations of FeSCN²⁺ complexes are measured at 447 nm to construct a standard   curve.  The unknown concentrations of FeSCN²⁺ ion in saliva samples are determined using the linear relationship between absorption and concentration as established by the standard curve.

            The results of this study show that the greater the exposure to the smoke is, the higher the concentration of thiocyanate ion is in saliva.  Since smokers are exposed to more tobacco smoke than nonsmokers, it seems that they should have an initial SCN^- concentration that is higher than nonsmokers and therefore a higher absorbance reading.  The saliva samples are collected from subjects with varying smoke exposure, including smokers, nonsmokers, nonsmokers who live and/or work with smokers, and a tobacco chewer.  The results are fairly consistent with other laboratory data, which shows that there is a linear relationship between exposure to tobacco smoke and concentration of the thiocyanate ion in the body.  

MATERIALS

            Spectronic Instruments Genesys 5 Spectrophotometer

            Quartz cuvettes

            0.0038 M Fe(NO₃)₃ � 9 H₂0 in 1 M aqueous HNO₃

            0.0002 M KSCN

            Tabletop centrifuge

            Other basic laboratory equipment

METHODS

             In conducting this lab, a standard curve is obtained using solutions listed in Table 1.  Keeping the amount of Fe³⁺ ions constant, as well as the total volume, and increasing only the amount of SCN^- ions added, an increasing amount of FeSCN²⁺ complex is formed in standard solutions.

Table I - Standard Curve Compositions

 Once the standard curve is obtained, the saliva samples are tested.  In order to prepare the samples for the experiment, they should be centrifuged at 3000 rpm for at least ten minutes.  The final solutions are obtained by mixing 0.5 ml of the resulting clear solution with 0.0019 M Fe(NO₃)₃ to give a total volume of 10 ml.  If thiocyanate ion is present, the clear solution becomes orange tinted.  The more thiocyanate ion that is present in the sample, the darker the orange color.  After all samples are prepared, the absorbances are measured at 447 nm against 0.0019 M Fe(NO₃)₃, which is used as a blank.  The results are presented in the following section.

RESULTS

            The absorbances of the standard solutions measured at 447 nm are presented in Table II.

Table II - Standard Curve Absorbances

The following graph is the standard curve, which was used to calculate the FeSCN²⁺ complex concentration of each saliva sample according to its absorbance reading.  That information was further used to calculate the SCN^- concentration in the original saliva samples.

Graph 1 - Standard Curve

                 The data for the saliva samples are presented in Table III.

Table III - Saliva Sample Data

             The FeSCN²⁺ concentration for each saliva sample is calculated from the absorbance values measured using the equation of a line for the standard curve (y = 0.0128x + 0.0011).  The initial SCN^- concentrations in saliva were then calculated using the formation constant for the FeSCN²⁺ complex formed in equation 2 as follows:

K_f = [FeSCN²⁺] / [Fe³⁺ - FeSCN²⁺][SCN^- - FeSCN²⁺]             Equation 3

The SCN^- concentration was then multiplied with the initial dilution factor to get the SCN^- concentration in the saliva.  The results are presented in the following table.

Table IV - Saliva Sample Results

 As is illustrated by this data, smokers and nonsmokers who live with smokers have higher absorbances at 447 nm, i.e., a higher concentration of thiocyanate ion, than nonsmokers with little exposure to tobacco smoke.  This data indicates that nonsmokers who are exposed to second hand smoke are in equal or more danger of ingesting the toxic HCN gas present in cigarette smoke.

            Graph II contains both the standard curve as well as all saliva samples.  This further illustrates the linear relationship between the amount of FeSCN²⁺ complex present and the absorbance reading.

Graph II � Standard Curve with Saliva Samples

            The graph seems to indicate that the last two samples have a complex amount that is much greater than the other samples, while in reality they are approximately twice the amount as the rest of the sample group. 

DISCUSSION

This study shows that it is possible to determine the amount of HCN present in tobacco smoke using a simple spectrophotometric technique. In this experiment, the absorbances of saliva samples treated with Fe³⁺ are used to qualitatively and quantitatively compare relative amounts of thiocyanate ion in smokers, nonsmokers, and nonsmokers who are exposed to second hand smoke.

Table IV illustrates the relationship between tobacco smoke exposure and thiocyante ion concentration.  The highest concentrations of thiocyanate ion belong to the donor who smokes the most and the donor who lives with two smokers.  They have the most exposure to the tobacco smoke and therefore, because of the linear relationship between the two variables, they have the highest thiocyanate ion concentrations.  As the sample's exposure to smoke increases, the thiocyanate ion concentration also increases.  In fact, with the exception of the nonsmoker who lives with two smokers, nonsmokers have lower thiocyanate concentrations than smokers.  The lowest thiocyanate ion concentration was surprisingly from the tobacco chewer.  It would seem that since the tobacco chewer has direct contact with the tobacco, that their thiocyanate ion concentration would be fairly high.  This is not the case though.  The thiocyanate ion is formed from the conversion of the HCN gas in the smoke produced from the tobacco not from the tobacco itself.  It can also be concluded from the direct relationship between smoke exposure and thiocyanate ion concentration that the tobacco chewer has the lowest amount of exposure to secondhand smoke. 

It is clear from the results of the saliva samples that there is a dangerous relationship between the relative amount of tobacco smoke and the amount of thiocyanate ion present in their body.  While most of the subjects in this study who are nonsmokers did not have a higher amount of thiocyanate ion as compared to the tobacco smoker, it is clear that the exposure to secondhand smoke can be just as harmful as the direct inhalation of it.  It is reported that if a nonsmoker is around a person who smokes a pack of cigarettes during the day, the nonsmoker�s exposure is comparable to his or her smoking half of that pack of cigarettes. (www.americanheart.org 4/11/01).  The results of this study suggests that the secondhand smoker�s exposure is even higher than previously thought and can be comparable to the total amount of exposure a smoker experiences.

  In the interpretation of the results, one should also consider factors such as other possible sources of cyanide.  It is known that certain foods, particularly leafy vegetables, some nuts, and certain drinks like beer contain cyanide. (Surgeon General, 1986, 202).  A more careful study requires monitoring the diet of each subject so that the possible competing sources of thiocyanate ion are known.  We intend to confirm the results of this study by taking into account the other possible sources of cyanide present in the subjects� diets in a follow-up experiment.  Another complication that must be considered is saliva activity.   It is suggested that the saliva flow rate at the time of sampling can influence the  thiocyanate concentration present in the sample (www.ucn.es 2/21/01). 

       HCN along with a number of other carcinogenic compounds can lead to many health problems.  As was mentioned earlier, the hazards of smoking include lung and heart disease.  They can also lead to mouth cancer, dental problems, asthma, and emphysema, as well as several other medical problems.  While the effects are greater in smokers, it is evident from the results of this study as well as many others, that secondhand smoke is detrimental to one�s health.

Works Cited

1.      C. Matheus and K.E. Van Holde; Biochemistry, The Benjamin/Cummings Company, 1990.

2.      Lahti, Markku, Vilpo, Juhani, and Hovinen, Jari, Spectrophotometric Determination of Thiocyanate in Human Saliva.  Journal of Chemical Education, Vol. 76, No. 9, September 1999.  pgs 1281-1282.

3.      Surgeon General, The Health Consequences of Involuntary Smoking: a report of the Surgeon General.  1986.  pg 202-203.

4.  Surgeon General, The Health Benefits of Smoking Cessation: a report of the Surgeon General.  1990.  pg. 35.

4.      Media Advisory on Secondhand Study Published in Journal of American Medical Association. http://www.americanheart.org/Whats_News/AHA_Science_Advisories/2HandSmoke.html

5.      American Lung Association Fact Sheet: Secondhand Smoke and Children. http://www.lungusa.org/tobacco/secondkids_factsheet.html

6.      Setting the Record Straight: Secondhand Smoke is A Preventable Health Risk. http://www.tobacco.org/Documents/9406EPA.html

7.      Clinical and Community Psychology. http://www.ucn.es/info/Psyap/231CAP/abstract/clinical4.html

Acknowledgment:

Special thanks to Dr. Feza Ozturk, Amie Touchette, Catharine Graham, Mike Melvin, Lindsay Braun, Gina Heinen, Aaron Krebs, Lyndon Mumm, Heather Heenan, and Lynn Hollenkamp for their contributions to this experiment.