Discussion
For
this experiment we were looking at cigars, chewing tobacco, and cigarettes,
and which causes the most damage to an organism. We believed that,
due to the amino acid tyrosine and the enzyme polyphenoloxidase found
in these products, all three will contribute to premature aging (Shi
C, et al., 2002). However, cigars will contribute more than the other
two. Furthermore, each sample will contain a certain number of sugars,
but cigars will contain the most. This sweet flavoring would explain
why cigars seem to be the most popular. All in all, cigars should
cause the more damage than cigarettes and chewing tobacco, based on
our sugar, photosynthetic, and pigment tests.
There are many different experiments that can be done to test for
various sugars. For our experiment, we chose to perform five of these
tests; Benedict's, Barfoed's, Selivanoff's, Bial's, and the Iodine
test. The results obtained from these tests were quite different from
what we expected. Two of the tests turned up negative (Benedict's
and Iodine). Because Benedict's test turned out negative, we ascertained
that neither of the three tobacco products contained a free aldehyde
or ketone group, therefore showing that these are not reducing sugars.
In addition, the Iodine test also turned up negative, implying that
no starch is contained within the cigar, cigarette and tobacco solutions.
However the remaining three tests generated positive results. We found
that although each sample contained the same number of sugars, they
contain different kinds of sugars. Bial's test came up positive for
all three samples. Cigars and chewing tobacco were found to have hexose
(or higher)-furanose, and cigarettes were found to contain pentose-furanose.
Barfoed's test resulted in a precipitate for all three samples. Cigars
had the most precipitate and therefore the largest amount of monosaccharides.
Chewing tobacco had the least amount of precipitate and therefore
contains the smallest amount of monosaccharides. Cigarettes were in
between cigars and chewing tobacco. Selivanoff's test was a bit harder
to record so all the findings are subjective. However, we found that
cigars contain disaccharide ketoses, cigarettes contain monosaccharide
ketoses, and chewing tobacco contains aldoses.
In summary, we found that cigarettes were non-reducing sugar hexose-furanose
rings with no starch, as well as containing monosaccharide ketoses.
Cigars were a non-reducing, non-starch containing penta-furanose rings
containing disaccharide ketoses. Finally, chewing tobacco was also
not a reducing sugar without any starch. As with the cigars, the chewing
tobacco also was a penta-furanose ring but with monosaccharide aldoses
instead (Figure 1).
From these results we cannot make a direct conclusion that cigars
are more popular because of the number of sugars they contain, although
cigars did contain the highest amount of monosaccharides. We can neither
make an accurate assumption as to whether cigars will aid in tooth
decay much more than the other tobacco products, although they did
contain the most precipitate from Barfoed's (which we believe indicated
the most monosaccharide content). Nonetheless, these tests did support
part of our hypothesis (where we asked which is more damaging) that
cigars, cigarettes, and chewing tobacco contain sugars and therefore
aid in tooth decay. As stated in the introduction, "the tobacco
sugars can contribute to tooth decay…[and] heighten the risk
of getting cavities" (Bachman, Richard E. et al., 2003).
Furthermore, based on the results found in the experiments with the
fast plants, we were not able to ascertain that cigars did the most
damage to the specimens. In order to discuss the photosynthetic part
of the experiment, we should first look at the trend in the growth
of the Wisconsin fast plants. Honestly, the plants that grew the highest
were the cigarettes, averaging at about 103 mm by the end of the treatment
period. For part of the treatment period though, specifically from
days 10 through 15, the chewing tobacco was the highest (Table 2).
Interestingly enough, the average growth of the control plant, without
any tobacco treatment, maintained a pretty linear fit, as did the
plants treated with cigars. This linear fit is expected for the control,
implying that the amount of distilled water used to treat the plant
was proportional to its daily growth (Figure 7). The interesting aspect
comes with the linear fit of the cigars. We believed that cigars would
cause the most damage by inhibiting the growth of the plant, and they
did. This inhibition along with the linear fit helps to show that
cigars are indeed hazardous to these plants, that the amount of cigar
solution the plant was treated with was directly proportional to its
low average growth heights. Yet this height was still a higher than
the averages of the control. This may be due to the process of planting
seeds. The control seeds may have been planted deeper in the dirt,
whereas the cigars may have been even a couple millimeters taller.
The average growths of the cigars and control, though, were relatively
close, which imply that this error may have occurred.
After our treatment period of the Wisconsin fast plants, we were able
to perform the paper chromatography test as well as the absorption
spectrum test as part of our photosynthetic experiments. Considering
that both plants and human cells are eukaryotic, we can apply results
found about our plants towards humans. Therefore, cigars would theoretically
also cause the most damage to humans. (Due to limitations in the lab,
we were only able to perform tests on plants and not any mammal.)
Following two trials of the paper chromatography test for each type
of tobacco-treated plant and the control, we were able to see a pattern
in the rate of flow as well as in the types of pigments present. We
believe that the pigments present in all the tobacco-treated plants
and the control are the same: chlorophyll a, chlorophyll b, carotenes,
and xanthophylls. We also noticed differences in the smearing patterns
of the pigments, but the main characteristics of the paper chromatography
test were the pigment jumps. While looking at the paper chromatogram
strips, one can clearly see a band of white (indicating that nothing
is there) between two yellow bands. Now, this can have two causes.
First, the two pigments separated by the pigment jump could just be
two different pigments all together. These two pigments would then
have been soluble at different distances. Our group believes that
this is the reason why the pigment jump occurred-there were two different
pigments. Also, pigment degradation may have also caused this pigment
jump. Because of enzyme inherently within each plant, snapping, or
breaking apart, of the pigment may have occurred, thereby showing
two yellow bands. The yellow bands may have initially been one band,
but its degradation will actually allow the different parts to be
soluble at different distances.
If this was the case, then another possible error for our experiment
occurred here. Our samples may not have been kept cold enough in order
to prevent this degradation. Further experimentation (by controlling
temperature and pH) would then be necessary to ascertain whether the
pigment jumping occurred because of this degradation.
With the paper chromatography test, we also calculated the rate of
flow for each of the tobacco products. This rate of flow is a ratio
of pigment distance over the solvent front (Krha et al., 2003). This
rate then tells one how soluble a certain pigment is at which distance.
Here, it would also be important to mention that this rate of flow
also depends on which solvent is being used. For example, the solvent
in our tests was petroleum ether, but the solvent used when a cigarette
or cigar is lit up or when chewing tobacco is chewed could very well
be salivary amylase. This difference in pH (from the buffers within
your mouth) and temperature may cause drastic differences in the rate
of flow. In addition, a positive correlation between the rate of flow
(smearing and distance traveled by the pigment) and the concentration
of that specific pigment may exist. A long pigment distance may indicate
a higher concentration of that pigment and vice versa. But, as stated
earlier, further experimentation will need to be done in order to
determine the validity of this statement. This experimentation may
involve specific concentrations of pigments that are held as a control.
Another part of our photosynthetic experimentation included an absorption
spectrum of each of the tobacco-treated plants and our control. This
absorption range indicates the best wavelengths at which photosynthesis
can occur in. Additionally, the optimum absorption for all the solutions
was at 430 nm with 1.140, 1.016, 0.920, and 0.872 for cigars, cigarettes,
chewing tobacco, and the control, respectively (Table 4). This 430
nm wavelength falls within the violet/blue light range, meaning that
this light is absorbed while green light is reflected. This gives
us another indication that our samples do contain the chlorophylls.
Yet, this very high absorbency at 430 nm in the blue region is abnormal.
These blue photons of light excite the electrons to a higher state
of energy (Freeman, 2002). These blue light photons can be accepted
only by carotene. Any other pigment (chlorophyll a or chlorophyll
b) will only reject this blue photon. Because of this high absorbance,
we are able to conclude that there is a higher concentration of carotene
within each of our samples.
In addition, another peak occurs for all the samples at 670 nm, with
the absorbencies of 0.607, 0.546, 0.493, and 0.475 for cigars, cigarettes,
chewing tobacco, and the control respectively (Table 4). This wavelength
falls within red light region of the visible spectrum. Peaks in this
red range represent that photosynthesis does occur within the plants,
however, due to the high concentration of carotene and conversely
the low concentration of chlorophyll a and chlorophyll b, not nearly
as much red light is absorbed as within the photosynthesis reactions
studied in class.
For the enzyme experimentation portion of our independent research
lab, we found that none of our tobacco leaves contained polyphenoloxidase
(PPO). We used a potato tuber as our control, and the presence of
PPO was immediately detected. Therefore, we know that our catechol
solution was not degraded. We did continue the environmental and heat
tests on PPO with each of our tobacco solutions, but they also have
shown that PPO was not present. Our short range of absorbencies for
each sample shows a flat line, which reveals that PPO was not present
also. Any increasing or decreasing in absorbencies may be due to the
catechol solution.
If PPO was found, we would have expected that a large concentration
of this enzyme would initiate the catalysis reactions a lot quicker,
thereby allowing the plant to metabolize quicker. By catalyzing these
oxidation reactions, the excess electrons lost from the oxidation
would theoretically go into the light reaction phase of photosynthesis.
Theoretically, this would speed up photosynthesis, thereby causing
premature aging.
But, we did not find the enzyme polyphenoloxidase in our tobacco samples.
This may be due to the fact that enzymes that were once within the
living tobacco plant have now degraded and denatured to the point
where they are not present within the tobacco leaves anymore. After
all, we were testing products that were once living, but have been
treated with so many chemicals that former enzymes are now not detectable.
Another explanation for no PPO is due to the fact that a "new
polyphenoloxidase (PPO) named PPO II was purified from tobacco (Nicotiana
tobacum) by using acetone powder, ammonium sulfate, and column chromatography"
(Shi C. et al, 2002). This PPO II may have different structural aspects
from the PPO we were trying to detect with the catechol solution.
Furthermore, lighting a cigarette or cigar may even spark or catalyze
certain reactions to occur within the tobacco leaves that we were
not able to detect because we did not "light up" with any
of our samples. Even the salivary amylase used when masticating chewing
tobacco would change the configuration of certain structures within
the reactions. Further experimentation, at this point, would be needed
to isolate the reason as to why PPO was not detected.
Nonetheless, due to the fact that PPO was not found using our enzyme
tests, we were not able to support our hypothesis. In essence, our
hypothesis was negated. Even the presence of sugars and pigments,
as well as the rate flows of the pigments, did not give us enough
support to determine whether or not cigars were more hazardous than
either cigarettes or chewing tobacco. The only trend that would support
our hypothesis is the fact that the Wisconsin fast plants which were
treated with cigars actually inhibited the growth of the plants.
There were some potential problems with the above results and inferences,
however. First, when performing the sugar tests, the concentration
of our samples may have been too low for there to be any significant
result. In addition, human error may have occurred in Selivanoff's
test because we may not have accurately predicted whether a certain
tobacco product was a ketose or an aldose. This is due to the timing
of the experiment. There is also the simple fact that we were forced
to use plants as our experimental subjects which raises many questions
about the accuracy of the data as pertains to humans. For example,
the plants absorbed the chemicals from the tobacco products through
their roots into their vascular system, whereas humans would take
in the chemicals through respiration, with the exception of chewing
tobacco, another problem in itself. There could be significant differences
between the amount of damage chewing tobacco might do if it could
be inhaled, for example.
Another weakness can be found in our uncertainty as to proper measurements.
We may have diluted the tobacco too much with the water given to the
fast plants, causing no visible effects where there should be some.
The always-present possibility of human error is also a site of prospective
weakness within the research. Finally, our research may be undermined
by the fact that some of our research and data can be dated as far
back as 1977.
