Reducing Sugars:
Using Glucosamine Supplements to Alter Bacterial DNA
John Goering
Goering 2
Natural Science Seminar
May 10, 2012
Abstract
Reducing sugars are a unique class of carbohydrates that are capable of being
oxidized in chemical reactions, thus causing other substances to be reduced.
Past research has shown that these sugars are capable of reacting with nucleic
acids, particularly in the form of bacterial plasmid DNA, to initiate chemical
changes such as nicks and cuts in the DNA strands.
The research described in
this paper specifically addresses the affects of a particular sugar, glucosamine,
on E. coli plasmid DNA.
It was found that not only can pure laboratory-grade
glucosamine initiate these changes, but also nutritional supplements intended to
enhance joint health, which contain glucosamine as a key ingredient.
In
addition, it is shown that buffers present in the reducing sugar-DNA reaction
solution can potentially inhibit some DNA alteration.
The effects of two
different pH buffers, tris and phosphate, are compared.
Tris buffer, which acts
as a free-radical inhibitor, is found to inhibit DNA damage to a small degree.
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Introduction
Researchers have long known that certain types of carbohydrate (sugar)
molecules are capable of reacting nonenzymatically with other biological
molecules – especially nucleic acids and proteins – to initiate chemical
changes.1
These sugars, known as reducing sugars, react with the amino
groups of biological molecules in a process called nonenzymatic browning.
The
reacting amino group is found either in the amino acids of proteins or the
nitrogenous bases of nucleic acids.
Reducing sugars were first discovered by food scientist L.C. Maillard in
1912.
Maillard noted that certain sugars could react with the amino groups of
proteins to form a stable, yellow-brown colored product.
This initial discovery
was of great interest to food scientists, as the process is at least partially
responsible for the browning and spoilage of fruits, vegetables, and other
foods.1,2
It has also become clear that reducing sugars are capable of reacting
with proteins and nucleic acids in living tissues, potentially causing damage.
Reducing sugars have been found to have significant effects on the
physiological functioning of living cells.
For example, glucose 6-phosphate has
been shown to have a mutagenic effect on the E. coli plasmid pBR322 in vivo.2
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Reducing sugars are also of great interest to some researchers for the
effects that they may have on human health.
Research suggests that even
glucose, the body’s most abundant sugar, may not be biologically inert as has
traditionally been assumed.
In fact, the effects of glucose on the proteins of
the body is now though to contribute to the aging process by causing
significant declines in the functioning of cells and tissues over time.3
Many
age-related declines in physiological function are due to the accumulation of
unrepaired genetic damage or mutations: DNA strand breaks, chromosomal
abnormalities, and errors in DNA replication, transcription, and repair.4
This project addresses the effects of a particular reducing sugar –
glucosamine – on the bacterial DNA plasmid pBR322, found in E. coli bacteria.
Previous research suggests that glucosamine is capable of causing single-strand
scission (“nicking”), double-stranded cleavage, and other damage.
In this
experiment, glucosamine samples were prepared from readily available over-thecounter joint-health supplements and reacted with pBR322, an E. coli DNA
plasmid.
Results obtained from reaction with these supplements are compared
with results from reaction with pure laboratory-grade glucosamine.
If
supplements intended for human consumption are capable of damaging
bacterial DNA, it may have implications for human health as well.
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Materials and Methods
There were two primary objectives for this experiment.
The first was to
determine the effects of various types of glucosamine on plasmid DNA.
Four
different glucosamine solutions were used, one of which was prepared using
laboratory-grade glucosamine powder, while the other three utilized readily
available over-the-counter nutritional supplements containing glucosamine.
The second objective was to determine the effects that different pH
buffers may have on the ability of glucosamine to react with DNA.
All reactions
were carried out in the presence of one of two different buffering solutions.
The buffers used were tris(hydroxymethyl)aminomethane hydrochloride (“Tris”)
and monobasic sodium phosphate (NaH2PO4).
inhibitor, while phosphate does not.
Tris functions as a free-radical
Because reducing sugars are believed to
exert their effects on nucleic acids by way of a free-radical mechanism, it is
hypothesized that reactions carried out in the presence of a tris buffer may
cause less DNA damage than those carried out in phosphate.
Reagents
Water: ultrapure deionized water was utilized to prepare all buffers and reaction
solutions for this experiment, in order to minimize the potential effects of
dissolved metals and other ions on the reaction.
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DNA: the DNA used in these experiments was E. coli plasmid pBR322 purchased
from Carolina Biological Supply.
The stock solution had a concentration of 0.1
µg/µL.
Phosphate buffer: prepared by dissolving 6.900 g of monobasic sodium
phosphate [NaH2PO4] in 100 mL of water, then using concentrated HCl to adjust
the pH to 7.2.
This stock solution was prepared at a concentration of 500 mM.
Tris buffer: prepared by dissolving 6.055 g of tris(hydroxymethyl)aminomethane
base [NH2C(CH2OH)3] in 100 mL of water, then adjusting to pH 7.2 with
concentrated HCl.
Final concentration was 500 mM.
Glucosamine samples:

Sigma glucosamine hydrochloride (0.1078 g in 1 mL of water for a 500
mM solution)

Supplement #1: The first supplement utilized was “Finest Natural
Glucosamine & Chondroitin.”
This supplement contained 500 mg of
glucosamine hydrochloride per tablet.
Tablet was crushed and dissolved
in water to yield a glucosamine HCl concentration of 500 mM.
The
tablet did contain some insoluble material, so the solution was
centrifuged and liquid pipetted from the top of the tube when adding to
reactions.

Supplement #2: The second supplement used was “Finest Natural
Glucosamine MSM.”
The active ingredient in these tablets was
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glucosamine sulfate rather than glucosamine HCl, and they also contained
MSM, or methylsulfonylmethane.
Again, the tablet was crushed and
dissolved and the resulting solution centrifuged to yield a solution
containing 500 mM glucosamine sulfate.

Supplement #3: The third supplement was “Joint Juice,” which is a
powder that is dissolved in water.
Because of this, it was entirely
soluble, but also contained dyes and flavorings.
The active ingredient
was glucosamine HCl (as in supplement #1), and was prepared at a
concentration of 500 mM.
Loading dye/stop solution: taken from a stock solution prepared by previous
students studying reducing sugar-DNA interactions.
Contains bromophenol blue
and xylene cyanol dissolved in water, glycerol, and Tris.
This solution served
two purposes: it was added to reaction solutions at specific time intervals to
“freeze” the reaction and preserve the DNA in its current state until gel
electrophoresis could be performed, and it also contained the loading dye
necessary to visualize the movement of the solution through the gel during
electrophoresis.
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Reaction Procedure
All reagents were measured using micropipettes and combined in 1.5-mL plastic
tubes.
For each glucosamine sample analyzed, four conditions were used: one
containing DNA, glucosamine, and tris buffer; one containing DNA, glucosamine,
and phosphate buffer; and two control reactions containing DNA and either tris
or phosphate buffer, but without any glucosamine present, to ensure that any
DNA damage effects are indeed the result of the presence of glucosamine.
Ultrapure water was added to each reaction to bring the total to 30 µL, in order
to dilute the reagents to concentrations known to work well as determined by previous
experimenters.
These tubes were briefly centrifuged to combine reagents, and
then placed in a 37°C water bath for the duration of the reaction.
Figure 1
below shows the contents of each of the four reactions prepared.
Figure 1: Contents of Glucosamine-DNA reactions
Reaction Condition
Glucosamine + Phosphate
Glucosamine + Tris
Phosphate Control
Tris Control
DNA
added
3
3
3
3
µL
µL
µL
µL
Glucosamine
solution
added
3 µL
3 µL
None
None
Tris buffer
added
Phosphate
buffer
added
None
3 µL
None
3 µL
3 µL
None
3 µL
None
Ultrapure
water
added
21
21
24
24
µL
µL
µL
µL
Total
vol.
30
30
30
30
µL
µL
µL
µL
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Immediately after combining the reagents, initial “reference” samples of
each of the four reaction mixtures were taken.
A 5 µL sample of each was
added to a clean tube, and 5 µL of stop solution was added to halt the
progress of the reaction.
electrophoresis.
These samples were refrigerated for later analysis by
The reaction tubes were then placed in a 37° C warm water
bath and the reaction allowed to progress.
reaction mixture after 24 hours had passed.
Samples were again taken from the
Gel electrophoresis was then
performed to allow comparison of the DNA before and after the reaction had
taken place.
It should be noted that while 0 and 24-hour samples were taken
for all 4 sets of reactions, additional 3 and 6-hour samples were also taken for
only the first set of reactions (those utilizing the laboratory glucosamine as a
reagent).
Earlier trials had shown that the DNA plasmids degraded rapidly after
about 24 hours, to the point that electrophoresis no longer yielded useful
results.
Thus, the reactions were concluded at 24 hours and no more samples
were taken.
Gel Electrophoresis Procedure
25 mL of 1% agarose in 1X TAE buffer was prepared by heating in a
microwave oven and poured into a gel casting tray with 12-well comb to
harden.
After the gel hardened, the comb was removed, and the gel placed
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into the electrophoresis apparatus, which was then filled with 1X TAE buffer to
cover the gel.
be analyzed.
Wells were loaded using 5 µL of desired reaction solutions to
Gels were electrophoresed at a constant voltage of approximately
100V for 30-45 minutes, or until loading dye reached 2⁄3 to 3⁄4 of the way to
end of the gel.
The gel was then stained with 0.5 µg/mL ethidium bromide,
while being lightly agitated, for about 5 minutes, then rinsed with deionized
water several times.
The gel was finally visualized under UV light and a
Polaroid picture taken for scanning and analysis.
Digital Image Analysis Procedure
To determine the effects of the various glucosamine samples in, the gel
photographs were digitally scanned in grayscale at 600 dpi, and saved in JPEG
format.
IMAL software (Image and Measurement Analysis Lab) is a scientific
image analysis product that was used to analyze the gels.
By using the “strip
densitometry” function to analyze each lane of the gel, it can be determined
how much DNA is found in each of the three possible forms.
Bacterial DNA plasmids are usually found in one of these three forms:
covalently closed circular (ccc, also known as “supercoiled”), nicked (or open
circular), or linear.
The supercoiled form is the most abundant form of pBR322
under normal conditions.
However, reducing sugars such as glucosamine are
known to be capable of inducing breakages in the strands of bacterial DNA,
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causing it to adopt the “nicked” conformation, and eventually a linear
conformation.
Thus, as the reaction progresses, it would be expected that
large amounts of nicked DNA would appear, and eventually linear DNA as well.
Figure 2 shows the various conformations of DNA.
Figure 2: Supercoiled vs. Nicked DNA
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Results
Reaction 1: Lab-grade Glucosamine (“control”)
CCC
Linear
Nicked
9 10 11 12
13 14 15 16
CCC
Nicked
1 2 3 4
Lane
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Time
Initial (0 hr)
Initial (0 hr)
Initial (0 hr)
Initial (0 hr)
3 hr
3 hr
3 hr
3 hr
6 hr
6 hr
6 hr
6 hr
24 hr
24 hr
24 hr
24 hr
5 6 7 8
Condition
Glucosamine + Tris
Glucosamine + Phosphate
Tris Control
Phosphate Control
Glucosamine + Tris
Glucosamine + Phosphate
Tris Control
Phosphate Control
Glucosamine + Tris
Glucosamine + Phosphate
Tris Control
Phosphate Control
Glucosamine + Tris
Glucosamine + Phosphate
Tris Control
Phosphate Control
% CCC
% Nicked % Linear
84.4%
15.6%
0.0%
81.6%
18.4%
0.0%
79.6%
20.4%
0.0%
84.7%
15.3%
0.0%
77.8%
22.2%
0.0%
59.0%
41.0%
0.0%
80.3%
19.7%
0.0%
79.6%
20.4%
0.0%
68.1%
31.9%
0.0%
45.1%
54.9%
0.0%
82.7%
17.3%
0.0%
79.6%
20.4%
0.0%
0.0%
59.9%
40.1%
0.0%
40.0%
60.0%
82.2%
17.8%
0.0%
71.3%
28.7%
0.0%
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"Control" Reaction
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
% CCC
% Nicked
% Linear
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Reaction 2: Finest Natural Glucosamine & Chondroitin Supplement
1 2 3 4
Lane
1
2
3
4
5
6
7
8
Time
Initial (0 hr)
Initial (0 hr)
Initial (0 hr)
Initial (0 hr)
24 hr
24 hr
24 hr
24 hr
5 6 7 8
Condition
Glucosamine + Phosphate
Glucosamine + Tris
Phosphate Control
Tris Control
Glucosamine + Phosphate
Glucosamine + Tris
Phosphate Control
Tris Control
% CCC % Nicked
48.9%
51.1%
51.0%
49.0%
100.0%
0.0%
95.1%
4.9%
0.0%
44.1%
0.0%
53.7%
81.3%
18.7%
79.2%
20.8%
% Linear
0.0%
0.0%
0.0%
0.0%
55.9%
46.3%
0.0%
0.0%
Supplement #1: "FN Glucosamine & Chondroitin"
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
% CCC
% Nicked
% Linear
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Reaction 3: Finest Natural Glucosamine MSM
1 2 3 4
Lane
1
2
3
4
5
6
7
8
Time
Initial (0 hr)
Initial (0 hr)
Initial (0 hr)
Initial (0 hr)
24 hr
24 hr
24 hr
24 hr
5 6 7 8
Condition
Glucosamine + Phosphate
Glucosamine + Tris
Phosphate Control
Tris Control
Glucosamine + Phosphate
Glucosamine + Tris
Phosphate Control
Tris Control
% CCC
N/A
N/A
N/A
N/A
0.0%
0.0%
74.7%
84.7%
% Nicked
N/A
N/A
N/A
N/A
40.6%
66.5%
25.3%
15.3%
% Linear
N/A
N/A
N/A
N/A
59.4%
33.5%
0.0%
0.0%
Glucosamine Supplement #2: "FN
Glucosamine MSM "
100%
80%
60%
% CCC
40%
% Nicked
20%
% Linear
0%
Glucosamine + Glucosamine + Phosphate
Phosphate (24)
Tris (24)
Control (24)
Tris Control
(24)
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Reaction 4: Joint Juice
1 2 3 4
Time (hrs)
Initial (0 hr)
Initial (0 hr)
Initial (0 hr)
Initial (0 hr)
24 hr
24 hr
24 hr
24 hr
Condition
Glucosamine + Phosphate
Glucosamine + Tris
Phosphate Control
Tris Control
Glucosamine + Phosphate
Glucosamine + Tris
Phosphate Control
Tris Control
%
%
% CCC
Nicked
Linear
88.7%
11.3%
0.0%
92.9%
7.1%
0.0%
100.0%
0.0%
0.0%
100.0%
0.0%
0.0%
0.0%
79.4%
20.6%
N/A
N/A
N/A
73.3%
26.7%
0.0%
83.0%
17.0%
0.0%
Glucosamine Supplement #3: "Joint Juice"
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Data Unavailable
Lane
1
2
3
4
5
6
7
8
5 6 7 8
% CCC
% Nicked
% Linear
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Discussion
The first question this experiment was intended to address was whether
or not the glucosamine contained in common nutritional supplements intended
for human consumption is capable of exerting the same type of effects on E.
coli plasmid pBR322 as pure glucosamine ordinarily used for laboratory
purposes.
This is clearly the case.
In each of the three reactions using the
supplements, significant DNA damage occurred in the solutions containing
glucosamine, while the control reactions had much lower levels of nicking.
The second major question addressed is whether tris buffer, as a freeradical inhibitor, is capable of mitigating the effects of glucosamine on DNA.
An examination of the results shows that in every case in which a comparison
can be made of the effects of both buffers, reactions carried out in tris buffer
showed less DNA damage (lower percentage of nicked/linear DNA) than those
carried out in phosphate buffer, after 24 hours of reaction time.
below shows this.
The graph
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Buffer Comparison
100.0%
90.0%
80.0%
70.0%
60.0%
50.0%
40.0%
30.0%
20.0%
10.0%
0.0%
% CCC
Reaction 1
Reaction 2
Reaction 3
Gluc. + Tris (24)
Gluc. + Phosphate (24)
Gluc. + Tris (24)
Gluc. + Phosphate (24)
Gluc. + Tris (24)
Gluc. + Phosphate (24)
Gluc. + Tris (24)
Gluc. + Phosphate (24)
% Nicked
% Linear
Reaction 4
A couple of other unexpected results were also noted.
For one, it is
clear that a low level of DNA damage occurred in the control reactions despite
the absence of glucosamine.
Additionally, a significant amount of DNA damage
appeared in the initial reaction samples, which were taken within a minute or
two of mixing the reagents.
This was particularly noticeable in with the reaction
containing “supplement #1.”
In this reaction, about 50% of the initial DNA was
nicked.
This could be because the glucosamine was able to react immediately
with the DNA, causing significant damage very quickly, or because the stop
solution in this case failed to completely stop the reaction.
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Acknowledgements
First and foremost, I would like to thank Dr. Gary Histand for providing the
guidance necessary to perform this project from beginning to end.
I would also
like to thank the Bethel College Chemistry Department for the provision of all
materials and equipment necessary to complete the project, as well as the
Biology Department for allowing the use of their DNA viewing equipment.
Lastly, I appreciate all of the support I have received from all my professors,
fellow students, and parents.
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References
1. Lee, A.; Cerami, A.
reducing sugars.
In vitro and in vivo reactions of nucleic acids with
Mutation Research, 1990, 185-191.
2. Lee, A. and Cerami, A.
Elevated glucose 6-phosphate levels are
associated with plasmid mutations in vivo.
Proc. Natl. Acad. Sci., USA.
Vol. 84, 1987, 8311-8314.
3. Lee, A.; Vlassara, H.; Brownlee, M. Glucose and Aging.
Scientific American,
1987, Vol. 256(5), 90-96.
4. Bucala, R.; Model, P.; Cerami, A.
Modification of DNA by reducing sugars:
A possible mechanism for nucleic acid aging and age-related dysfunction
in gene expression.
Proc. Natl. Acad. Sci., USA. Vol. 81, 1984, 105-109.
5. Image: http://users.wmin.ac.uk/~redwayk/lectures/images/super.gif