Name: K***** K*** ID#: *********

Lab Partner: V******* S****** Date: Wednesday 22nd September, 2010

Course Code: BIOL 2363 –Metabolism

Title of Lab: Assay of Tissue Glycogen

Results:

Table 1: Calibration curve for Glucose

Tube # 1 2 3 4 5 6 7
mL of stock 0.0 0.2 0.2 0.4 0.6 0.6 1.0
glucose
mL of water 1.0 0.8 0.8 0.6 0.4 0.4 0.0
mL enzyme 2.5 2.5 2.5 2.5 2.5 2.5 2.5
cocktail
A450nm 0 0.190 0.186 0.406 0.581 0.581 0.597
mol of glucose 0 0.040 0.040 0.080 0.120 0.120 0.200

Table 2: Assay of Tissue Glycogen from Fed tissue

Fed Tissue HEART MUSCLE KIDNEY LIVER
Mass of Tissue (g) 5.8 40 12.4 40
Final volume (mL) 12 106 32 107

Aliquot size mL 0.5 1.0 0.3 0.5 0.5 1.0 0.3 0.5
A450nm Sample i 0.060 0.09 0.191 0.124 0.015 0.02 0.080 0.199
7 6
Micromoles of glucose 0.010 0.02 0.045 0.025 0.002 0.00 0.018 0.045
0 6
Average glucose content (per 0.020 0.100 0.005 0.075
mL)
Concentration of glucose mol 2.59 16.6 0.806 50.2
(per g of tissue)
Mass of glycogen per 100g of 0.042 0.268 0.013 0.812
tissue

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Table 3: Assay of Tissue Glycogen from Fasted tissue
Fasted Tissue HEART MUSCLE KIDNEY LIVER
Mass of Tissue (g) 5.8 40 11.4 40
Final volume (mL) 11 106 30 106

Aliquot size mL 0.5 1.0 0.3 0.5 0.5 1.0 0.3 0.5
A450nm Sample i 0.092 0.19 0.034 0.044 0.036 0.07 0.063 0.090
9 4
Micromoles of glucose 0.020 0.04 0.006 0.008 0.006 0.01 0.010 0.020
5 4
Average glucose content (per 0.043 0.018 0.013 0.037
mL)
Concentration of glucose (per g 5.10 2.98 2.14 2.45
of tissue)
Mass of glycogen per 100g of 0.083 0.048 0.035 0.040
tissue

Calculations:

 Determining mmol of Glucose for Table1:

E.g. using test tube # 2

Stock glucose solution (0.2 μmoles/mL) ∴ 0.2mL will contain 0.2 ×0.2 = 0.04 μmoles glucose

 Concentration of glucose μmoles/g of tissue

E.g. using Fed Heart Tissue

Mass= 5.8g ; Final Volume= 12mL

Aliquots used: 0.5mL & 1.0mL

From Calibration Curve, 0.5mL contains 0.01 μmoles glucose ∴ 1mL will contain 0.02 μmol glucose
1mL contained 0.02 μmoles glucose according to curve

Hence, there is an average of 0.02 μmoles of glucose per mL of fed heart tissue.

In 5mL there will be 0.02 x 5 = 0.1 μmoles and in 25mL: 0.1 x 25 = 2.5 μmoles glucose

This is from the original 2mL sample of homogenate.

2.5mL μmoles in 2mL and final volume was12mL ∴ in 12mL there was (2.5/2) x 12= 15 μmoles

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Total mass of organ = 5.8g

Μmoles/g = 15/5.8 = 2.586 μmoles/g

 Mass (in grams) of glycogen per 100g of tissue (fed heart)

Mr. of glycosidic tissue = 162

2.586× 10−3
∴ x 162= 4.19×10−4 g of glycogen per gram of tissue.
1000

Hence in 100g there will be 0.042g glycogen.

Discussion:

A glycogen molecule consists of entirely glucose units linked together by α1-4 and α1-6
linkages [ CITATION Ham05 \l 11273 ].

[ CITATION Kin10 \l 11273 ]

Glycogen is stored mainly by the liver and skeletal muscle as an energy reserve. The role of
stored glycogen in muscle is to provide a source of energy upon extensive muscle contraction.
Glycogen in the liver is used to maintain blood glucose levels [ CITATION Nel \l 11273 ].

[ CITATION Lod99 \l 11273 ]

After a meal, glucose levels in the blood rise and liver cells link the excess glucose molecules
into long, branching chains of glycogen via condensation reactions [ CITATION Whi08 \l 11273 ],
hence decreasing the blood glucose concentration but increasing the amount of glycogen.

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 [ CITATION Nel \l 11273 ]

In the liver tissue of the fed rat, the concentration of glucose was 50.2μmol/g and the mass
of glycogen was 0.812g per 100g. In the liver tissue of the fasted rat, the glucose concentration was
2.45 μmol/g whereas the glycogen mass was 0.040g per 100g. The glucose concentration present in
the liver of the fasted rat was much lower than that of the fed rat or a rat under “normal
conditions”.

When blood glucose levels fall, glucagon acts on the liver to stimulate glycogen breakdown
to glucose which is released into the bloodstream to raise the blood glucose levels once again
[ CITATION Ham05 \l 11273 ].

Hence the lower levels of glycogen in the fasted rat compared to that of the fed rat. The low
blood glucose in the fasted rat would have allowed for glycogenlysis to occur resulting in the
decrease in glycogen concentration and increase in glucose concentration. In the fed rat however,
excess glucose would have triggered insulin which stimulates glycogen synthesis.

In the muscle tissue of the fed rat, the concentration of glucose was 16.6μmol/g and the
mass of glycogen was 0.268g per 100g. In the muscle tissue of the fasted rat, the glucose
concentration was 2.98 μmol/g whereas the glycogen mass was 0.048g per 100g. The glucose
concentration present in the muscle of the fasted rat was much lower than that of the fed.

Muscle cells do not store glycogen like the liver; they contain less glycogen per gram of
tissue. The glycogen present in the muscle is used to produce energy rapidly and due to the absence
of the enzyme glucose-6-phosphatase, the glucose-6-phosphate enters glycolysis and is oxidized to
yield energy for muscle contraction. The absence of the enzyme glucose-6-phosphatase prevents
the glucose obtained from entering into the blood stream and affecting blood glucose concentration
[ CITATION Nel \l 11273 ].

As a result, when the fasted rat utilized its glycogen storage in the muscles by contracting its
muscles, the amount of glycogen present decreased. In the fed rat however, as its glycogen
concentration was depleted, it was replenished since the rat was allowed to eat food. This is why
the concentrations of both glucose and glycogen were greater in the fed rat.

In the kidney tissue of the fed rat, the concentration of glucose was 0.806μmol/g and the
mass of glycogen was 0.013g per 100g. In the kidney tissue of the fasted rat, the glucose

4
concentration was 2.14μmol/g whereas the glycogen mass was 0.035g per 100g. The glucose
concentration present in the kidney of the fasted rat was greater than that of the fed rat.

The kidney stores very little glycogen, however it uses glucose taken up from the blood as a
source of energy. Glucose entering the kidney is always reabsorbed into the bloodstream. It is not
excreted via urea in healthy patients.

Due to the low levels of blood glucose in the fasted rat, its glycogen stores were being
broken down into glucose hence decreasing its glycogen concentration and increasing its glucose
concentration. The kidneys use glucose as a source of energy as well as reabsorb glucose into the
bloodstream. This would explain the higher level of glucose present in the fasted rat compared to
the fed rat. Additionally, the concentration of glucose is higher than the concentration of glycogen in
the fed rat. This could be as a result of the rat just finished having a meal and glycogen synthesis
was still taking place.

In the heart tissue of the fed rat, the concentration of glucose was 50.2μmol/g and the mass
of glycogen was 0.812g per 100g. In the heart tissue of the fasted rat, the glucose concentration was
2.45μmol/g whereas the glycogen mass was 0.040g per 100g. The glucose concentration present in
the heart of the fasted rat was greater than that of the fed rat.

Heart muscle is continuously active and has a completely aerobic metabolism at all times. It
uses mainly free fatty acids, but also some glucose and ketone bodies taken up from the blood as
sources of energy. Those fuels are oxidized via the citric acid cycle and oxidative phosphorylation to
generate ATP. It does not store glycogen in large amounts [ CITATION Nel \l 11273 ].

Since the heart is not a major store of glycogen, the fasted rat’s heart’s supply of glycogen
would have been utilized in a very short space of time. Additionally, since its blood glucose level
was low, the heart would not have been able to use much glucose as a source of energy explaining
why the levels of glucose and glycogen found in the heart tissue were so low. The fed rat however,
would have had a high blood glucose level and its heart would have been able to use glucose as a
source of energy, hence the high levels of glucose found.

Additional Discussion:

1) Role of Glycogen in liver: Glycogen acts as a storage molecule in the liver. As blood glucose
levels rise, the pancreas secretes insulin. When the glucose reaches the liver cells, insulin
acts on the hepatocytes and stimulates the release of glycogen synthase. Glucose molecules
continue bind to form glycogen chains. When blood glucose levels drop, the glycogen in the
liver can be broken down to form glucose to be released into the blood stream [ CITATION
Ham05 \l 11273 ].

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 Role of Glycogen in muscle: Glycogen stores glucose in the muscle tissue. It is unable to pass
glucose into the bloodstream due to the absence of glucose-6-phosphotase, hence the
glucose obtained in the muscle is used for internal muscle use [ CITATION Nel \l 11273 ].

Role of Glycogen in kidney: Glycogen acts as a storage molecule of glucose in the kidney as a
source of energy. The kidney, however stores very little glycogen.

Role of Glycogen in heart: Glycogen is used as a source of energy; however the heart only
stores glycogen in very little amounts. It uses mainly free fatty acids, glucose and ketone
bodies taken up from the blood as sources of energy [ CITATION Nel \l 11273 ].

2) Hepatocytes store glycogen equivalent to a glucose concentration of 0.4M. The actual

concentration of glycogen which is insoluble is about 0.01μM. If the cytosol contained 0.4M
glucose, the osmolarity of the cell would be elevated leading to entry of water via osmosis
that would eventually rupture the cell. Additionally the free-energy change for glucose
uptake into cells against very high concentration gradients would be extremely large
[ CITATION Nel \l 11273 ]. Storing glucose as glycogen eliminates these problems.

3) ATP

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References

Hames, David, and Nigel Hooper. 2005. Instant Notes Biochemistry 3/e. New York: Taylor & Francis
Group,

King, Michael W. The Medical Biochemistry Page. March 24, 2010.

http://themedicalbiochemistrypage.org/carbohydrates.html (accessed September 21, 2010).

Lodish, Harvey, Arnold Berk, Lawerence Zipersky, Paul Matsudiara, David Baltimore, and James
Darnell. 2007. Molecular Cell Biology. W.H. Freeman,

Nelson, David L, and Michael M Cox. 2008. Lehninger Principles of Biochemistry. United States of
America: W.H. Freeman and Company,

Whitney, Elle, and Sharon Rady Rolfes. 2008. Understanding Nutrition 11th Edition. California:
Wadsworth Publishing,