Reducing Sugars:Using Glucosamine Supplements to Alter Bacterial DNA

John Goering

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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

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

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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-the-counter 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.

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

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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 (Na H 2P O 4). Tris

functions as a free-radical inhibitor, while phosphate does not. 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.

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.

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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

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

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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.

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

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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

DNA added

Glucos-amine

solution added

Tris buffer added

Phosphate buffer

added

Ultrapure water added

Totalvol.

Glucosamine + Phosphate

3 µL 3 µL None 3 µL 21 µL 30 µL

Glucosamine + Tris 3 µL 3 µL 3 µL None 21 µL 30 µLPhosphate Control 3 µL None None 3 µL 24 µL 30 µLTris Control 3 µL None 3 µL None 24 µL 30 µL

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. These samples were refrigerated for later analysis

by electrophoresis. The reaction tubes were then placed in a 37° C warm

water bath and the reaction allowed to progress. Samples were again taken

from the reaction mixture after 24 hours had passed. 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).

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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

into the electrophoresis apparatus, which was then filled with 1X TAE buffer

to cover the gel. Wells were loaded using 5 µL of desired reaction solutions

to be analyzed. Gels were electrophoresed at a constant voltage of

approximately 100V for 30-45 minutes, or until loading dye reached 23 to 34 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

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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, 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

CCCLinearNicked

CCCNicked

1 2 3 4 5 6 7 8

9 10 11 12 13 14 15 16

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Results

Reaction 1: Lab-grade Glucosamine (“control”)

Lane Time Condition % CCC% Nicked % Linear

1 Initial (0 hr) Glucosamine + Tris 84.4% 15.6% 0.0%

2 Initial (0 hr)Glucosamine + Phosphate 81.6% 18.4% 0.0%

3 Initial (0 hr) Tris Control 79.6% 20.4% 0.0%4 Initial (0 hr) Phosphate Control 84.7% 15.3% 0.0%5 3 hr Glucosamine + Tris 77.8% 22.2% 0.0%

6 3 hrGlucosamine + Phosphate 59.0% 41.0% 0.0%

7 3 hr Tris Control 80.3% 19.7% 0.0%8 3 hr Phosphate Control 79.6% 20.4% 0.0%9 6 hr Glucosamine + Tris 68.1% 31.9% 0.0%

10 6 hrGlucosamine + Phosphate 45.1% 54.9% 0.0%

11 6 hr Tris Control 82.7% 17.3% 0.0%12 6 hr Phosphate Control 79.6% 20.4% 0.0%13 24 hr Glucosamine + Tris 0.0% 59.9% 40.1%

14 24 hrGlucosamine + Phosphate 0.0% 40.0% 60.0%

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15 24 hr Tris Control 82.2% 17.8% 0.0%16 24 hr Phosphate Control 71.3% 28.7% 0.0%

Glucosam

ine + Tr

is (0)

Glucosam

ine + Tr

is (3)

Glucosam

ine + Tr

is (6)

Glucosam

ine + Tr

is (24)

Glucosam

ine + Phosp

hate (0

)

Glucosam

ine + Phosp

hate (3

)

Glucosam

ine + Phosp

hate (6

)

Glucosam

ine + Phosp

hate (2

4)

Tris C

ontrol (0

)

Tris C

ontrol (3

)

Tris C

ontrol (6

)

Tris C

ontrol (2

4)

Phosphate

Control (0

)

Phosphate

Control (3

)

Phosphate

Control (6

)

Phosphate

Control (2

4)0%

20%

40%

60%

80%

100%

"Control" Reaction

% CCC% Nicked% Linear

1 2 3 4 5 6 7 8

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Reaction 2: Finest Natural Glucosamine & Chondroitin Supplement

Lane Time Condition % CCC%

Nicked % Linear

1 Initial (0 hr)Glucosamine + Phosphate 48.9% 51.1% 0.0%

2 Initial (0 hr) Glucosamine + Tris 51.0% 49.0% 0.0%3 Initial (0 hr) Phosphate Control 100.0% 0.0% 0.0%4 Initial (0 hr) Tris Control 95.1% 4.9% 0.0%

5 24 hrGlucosamine + Phosphate 0.0% 44.1% 55.9%

6 24 hr Glucosamine + Tris 0.0% 53.7% 46.3%7 24 hr Phosphate Control 81.3% 18.7% 0.0%8 24 hr Tris Control 79.2% 20.8% 0.0%

1 2 3 4 5 6 7 8

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Glucosam

ine + Phosp

hate (0

)

Glucosam

ine + Phosp

hate (2

4)

Glucosam

ine + Tr

is (0)

Glucosam

ine + Tr

is (24)

Phosphate

Control (0

)

Phosphate

Control (2

4)

Tris C

ontrol (0

)

Tris C

ontrol (2

4)0%

20%40%60%80%

100%

Supplement #1: "FN Glucosamine & Chondroitin"

% CCC% Nicked% Linear

Reaction 3: Finest Natural Glucosamine MSM

Lane Time Condition % CCC% Nicked % Linear

1 Initial (0 hr)Glucosamine + Phosphate N/A N/A N/A

2 Initial (0 hr) Glucosamine + Tris N/A N/A N/A3 Initial (0 hr) Phosphate Control N/A N/A N/A4 Initial (0 hr) Tris Control N/A N/A N/A

5 24 hrGlucosamine + Phosphate 0.0% 40.6% 59.4%

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6 24 hr Glucosamine + Tris 0.0% 66.5% 33.5%7 24 hr Phosphate Control 74.7% 25.3% 0.0%8 24 hr Tris Control 84.7% 15.3% 0.0%

Glucosamine + Phosphate (24)

Glucosamine + Tris (24)

Phosphate Control (24)

Tris Control (24)

0%

20%

40%

60%

80%

100%

Glucosamine Supplement #2: "FN Glucosamine MSM "

% CCC% Nicked% Linear

1 2 3 4 5 6 7 8

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Reaction 4: Joint Juice

Lane Time (hrs) Condition % CCC% Nicked

% Linear

1 Initial (0 hr)Glucosamine + Phosphate 88.7% 11.3% 0.0%

2 Initial (0 hr) Glucosamine + Tris 92.9% 7.1% 0.0%3 Initial (0 hr) Phosphate Control 100.0% 0.0% 0.0%4 Initial (0 hr) Tris Control 100.0% 0.0% 0.0%

5 24 hrGlucosamine + Phosphate 0.0% 79.4% 20.6%

6 24 hr Glucosamine + Tris N/A N/A N/A7 24 hr Phosphate Control 73.3% 26.7% 0.0%8 24 hr Tris Control 83.0% 17.0% 0.0%

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Glucosam

ine + Phosp

hate (0

)

Glucosam

ine + Phosp

hate (2

4)

Glucosam

ine + Tr

is (0)

Glucosam

ine + Tr

is (24)

Phosphate

Control (0

)

Phosphate

Control (2

4)

Tris C

ontrol (0

)

Tris C

ontrol (2

4)0%

20%40%60%80%

100%

Glucosamine Supplement #3: "Joint Juice"

% CCC% Nicked% Linear

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 free-

radical 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

Data

Una

vaila

ble

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DNA) than those carried out in phosphate buffer, after 24 hours of reaction

time. The graph below shows this.

Gluc

. + P

hosp

hate

(24)

Gluc

. + T

ris (2

4)

Gluc

. + P

hosp

hate

(24)

Gluc

. + T

ris (2

4)

Gluc

. + P

hosp

hate

(24)

Gluc

. + T

ris (2

4)

Gluc

. + P

hosp

hate

(24)

Gluc

. + T

ris (2

4)Reaction 1 Reaction 2 Reaction 3 Reaction 4

0.0%10.0%20.0%30.0%40.0%50.0%60.0%70.0%80.0%90.0%

100.0%

Buffer Comparison

% CCC% Nicked% Linear

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. In vitro and in vivo reactions of nucleic acids with reducing sugars. 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