Clinical Post Lab 2

8/13/2019 Clinical Post Lab 2

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Figure 1. LDH isozyme banding patterns of heart, liver, muscle serum obtained from

polyacrylamide gel electrophoresis (PAGE)


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LDH gel: heart, liver muscle, serum, ladder, ladder, serum, muscle, liver, heart.

Figure 2 Results of polyacrylamide gel electrophoresis of serum proteins


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Heart (lane1), liver (lane2), muscle (lane3), serum (lane4), the dye (lane 5) the ladder (lane 6),

heart (lane7), liver (lane, muscle (lane9, serum (lane10)

DISCUSSION:


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Lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27; LD) is an enzyme that

catalyzes the reversible reaction in which pyruvate and lactate are inter-converted (Kory, Susan

1993).

(Anderson S, Cockayne S. 2003.)

This enzyme is found in cells of many body tissues, it is found in the heart, liver, kidneys, skeletal

muscle, brain, red blood cells, and lungs. (Gennady P. Manchenko. 2003)

Due to the fact that LDH is present in almost all tissues in the body, the LDH test is used its in

order to detect if there are any alterations in tissues and it is also used to help diagnosis heart

attack, anemia, and liver disease. (Pagana, Deska 1998).

There are five different isoenzymes of LDH these are LDH1 LDH2 LDH3 LDH 4 andLDH5,

these different isoenzymes are separated based on their electrophoretic mobility (Kory, Susan

1993).

In LDH there are two different types of polypeptide chains, these are known as M and H, M for

skeletal muscle and H for the heart muscle. This H and H can be combined into the tetramer of

LDH in 5 ways. Each different combination of subunits represents a distinct LDH isoenzyme.

The electrophoretic mobilities of LDH are LDH 1 > LDH 2 > LDH 3 > LDH 4 > LDH 5 since

the H polypeptide has more acidic amino acid residues than the M. This is displayed below:

(Saito, Oohashi 1964).


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(Saito, . Oohashi. 1964. )

Discontinuous (disc) electrophoresis utilizing polyacrylamide as the supporting medium has been

claimed as one of the most effective methods for the separation of ionic components. As the

name indicates, it employs discontinuous (multiphasic) buffers varying in chemical composition

and properties on electrode wells and gels. This is the specific method employed for LDH

isoenzymes separation. (Mc Kenzie, Henderson. 1983.)

After electrophoresis the LDH isoenzymes can be visualized by an activity staining process

where the product of the enzymic reaction is a water insoluble stain precipitating in the gel where

the LDH proteins are located. The following reactions basically show the principle being the

staining technique: (Bhagavan 1992).

1) Lactate + NAD+

Pyruvate + NADH + H+

2) NADH + PMS NAD+ + PMS-H (PMS - Phenazine methosulfate)

3) PMS-H + TNBT PMS + TNBT-Formazan. (TNBT- Tetranitroblue tetrazolium)


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It is the TNBT-Formazan which is intensely colored that localizes in the electrophoretic zones of

LDH activity. The coloration that occurs directly indicates the amount of particular LDH

isoenzyme that is present.(Mc Kenzie, Henderson. 1983.)

The normal level of total lactate dehydrogenase is 105 - 333 IU/L (international units per liter).

If this level is elevated it may indicate the following:

Cerebrovascular accident (CVA, stroke) Heart attack Hemolytic anemia Low blood pressure Infectious mononucleosis Blood deficiency (intestinal ischemia) Liver disease (for example,hepatitis) Muscle injury Muscular dystrophy New abnormal tissue formation (neoplastic) states Pancreatitis

Tissue death (pulmonary infarction)

(Arbeloff, Armitage 2004).
http://www.nlm.nih.gov/medlineplus/ency/article/000726.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000195.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000571.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000591.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000205.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/001154.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/001190.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/001144.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/001144.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/001190.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/001154.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000205.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000591.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000571.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000195.htmhttp://www.nlm.nih.gov/medlineplus/ency/article/000726.htm


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Sucrose was used when making up each solution, the reason for this itsso that the solution willbe dense when it is running on the gel, its avoids the solution from coming off the gel and giving

results which are inaccurate. It is used to lyse the cells in the supernatants and to thus release the

contents of the cells including their enzymes.

An advantage of using polyacrylamide is the degree of cross linking of the gel that is present.

Also the gel hardness is a factor that plays a role in the high resolution of bands obtained. Hard

gels (12-20% acrylamide) retard the migration of large molecules more than they do small ones

while in loose gels (4-8% acrylamide), high molecular weight molecules migrate much farther

down the gel and, in some instances, can move right out of the matrix. (Kusa M. 1966. ).

From the above figure it can be seen that the bands were separated this was done by SDS

PAGE. The reason that the bands were seen in this order is that at pH 7 the H protomer is more

negative than the M protomer .Due to this the isoenzymes which contain H in a higher

proportion will have a net change which is more negative and due to this it would move faster

towards the anode .( Gennady P. Manchenko. 2003.)

LDH1 has four H subunits as a result it will move fastest towards the anode so it is therefore

was located closest to the anode.

LDH2 has three H subunits and it had one M subunit as a result it will move slower than LDH1

therefore it was located the second closest to the anode,


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LDH3 has an equal amount of M and H subunits, i.e. 2M and 2H; this was seen third furthest

from the anode so it therefore moves slower than LDH1 and LDH2

LDH4 was the fourth furthest from the anode the reason for this is that it has 3 M and 1 H so it

therefore moves slower than LDH1 LAH2 LAH3

LDH5 furthest from the anode but closest to the cathode this does not contain any H subunits.

The size of the different isoenzymes also had an impact on their movement down the gel .The net

change had a greater impact on this.

(Gennady P. Manchenko. 2003.).

Theoretically LDH1 and LDH2 are found predominately in the heart red cells and kidney. In this

experiment the Heart was added to well one from the results it can be seen that there were four

distinct bands, these bands show LDH1 LDH2 LDH3 and LDH 4(these were found in high

quantities in the heart) .from the figure of the gel obtained we can say that LDH1 was the first to

separate as compared to the others and due to this it is the furthest away from the point of

application, LDH 4 travelled the slowest and was closest to the application point. LDH5 was not

seen in this lane because isoenzyme LDH 5 is made of only the 4M genes, this can be seen in the

table below .LDH1 LDH2 LDH3 and LDH 4 contained the H gene.


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Liver was added to well two, a very thick band was observed near the point of application this

band can be isoenzyme LDH-5 with some LDH-4.The reason for this is because LDH4 and

LDH5 enzyme is high in the liver as compared to the other tissues especially the heart, there

were small amounts of LDH1 LDH2 and LDH3 obtained.

Theoretically LDH4 and LDH5 should also be predominant in some skeletal muscles. In well

three muscle was added, four bands were observed just as in the heart ,(LDH1 LDH2 LDH3 and

LDH4) however the bands for the heart was much deeper the reason for this is that the

isoenzymes were more concentrated in the heart . One reason in which the heart would have

deeper bands as compared to the muscle is that these samples were obtained from a rat, this

animal is very small in size and due to this its heart rate would be very high, as a result the heart

will be more active compared to the other organs in the body due to this more energy would be

needed for it to function thus the higher concentration of isoenzyme would supply this need for

more energy. From the results LDH4 had a darker band as compared to the rest; this proved thatLDH4 was predominant in skeletal muscle.

The serum was added to well four no distinct bands were seen , only the dye was shown .The

results appeared as a smear .This is because since LDH is present in almost all tissues, its

increase in serum is non-specific. But this suggests that no lactate dehydrogenase was present in


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the serum. Another caused is that the serum used in this experiment was diluted and probably

due to dilution if it had any small amount of lactate dehydrogenase it would not show up in the

gel.

Theoretically the LDH isoenzyme in healthy adult serum has the order as follows: (Bhagavan

1992)

LDH2LDH1LDH3LDH4LDH5.

(Bhagavan 1992)

The table below shows abnormalities in LDH isoenzyme pattern in serum where anodic

increases refer to LDH1 and LDH2 and cathodic increases refer to LDH4 and LDH5. (Mc

Kenzie, Henderson 1983).


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For the LDH stain the mechanism is as follows:

Substrate (red) NAD+ PMS (red) tetrazoliun salt

(Yellow soluble)

Enzyme

Substrate (ox) NADH PMS (ox) tetrazolium salt

(Red)

(Clinical Biochemistry Manual, 2012)

Serum electrophoris was done in the second part of this experiment in order to observe the

normal banding pattern in serum proteins.

Serum electrophoresis separates proteins based on their different electrical charges. The bands

produced are visualized and quantified using densitometry. (Anderson, Cockayne 2003) Serum

Protein electrophoresis (SPE) is a common screening test used to evaluate many diseaseprocesses. In this type of electrophoresis, proteins are separated based on their relative mobilities

in an electric field. (Mc Clatchey 2001).

Blue precipitate of formazan


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(Pagana, Deska 1998).

The results obtained is seen in figure 2 , different bands were observed because the gel was

stained with coomassie blue .Whether there is a small concentration or large it is expected that

all the isoenzymes will be present on the bands .

It is expected that LDH 5 will be the closest from the point of application and LDH1 will be the

furthest from the application point. LDH2 will be the second closest from application .LDH3 will

the third closest and then LDH4.

Below shows the order in which the sample was placed

heart (lane1 )-liver (lane2)-muscle (lane3)-serum (lane4),-dye(lane 5) - ladder (lane 6), heart

(lane7 ), liver (lane8) , muscle (lane9,) serum (lane10)


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Two different dyes were used for both experiments, these were, nitroblue tetrazolium and

coomassie blue. For the protein gel the better dye is coomassie blue (R-250) the reason for this is

that coomassie blue is able to detect up to 0.1 microgram of protein.

Coomassie blue stained the proteins by binding to them .When this was done the proteins got an

overall negative charge .This due did not denature the proteins; it allowed the protein to be

separated by polyacrylamide gel electrophoresis. The size and charge of the protein had an

influence in the way in which it moved throughout the gel.

From the results obtained it can be seen that there was a normal banding pattern for the serum in

both cases it can therefore be stated that there was no d isease in the patient, If a thick band was

obtained letssay at LDH1 this will suggest that the patient may have heart problems thus there

is an increase in the amount of LDH1 isoenzyme.

The muscle heart and liver were electrophoresed in order to determine the total amount of

protein contained. The serum was somewhat consistent with the BSA molecular ladder.Where it

did not line up some experimental errors may have occured . Serum contained the most protein;

this is because blood is the major transport medium around the body and contains a large

percentage of proteins.

This muscle had the most protein as compared to the liver and muscle the reason for this its

because muscle consists of myosin and actin which provide the major function for movement.

(Bhagavan N. 1992.)

Liver contained less protein than the muscle; this is due to the fact that the liver is responsible

for the regulation of many physiological conditions of the body. For example; blood glucose

levels, protein levels, break down of hormones etc.

The heart had the least amount of protein is a specialised tissue and the amount of proteins being

made are constant but are always being expressed.

Some errors may have occurred in the experiment because most of the bands did not line up with

the molecular ladder.


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REFERENCES:

Anderson S, Cockayne S. 2003. Clinical Chemistry: Concepts and Applications. Illustrated Edition. Mc

Graw Hill Professional. USA.

Bangert, Marshall. 2004. Clinical Biochemistry. 5th

edition. Mosley Publishers. UK

Bhagavan N. 1992. Medical Biochemistry. 3rd

edition. Jones and Bartlett Publishers. UK

Gennady P. Manchenko. 2003. Handbook of Detection of Enzymes on Electrophoretic Gels. CRC Press.

USA.

Kory, Susan. 1993.Clinical Chemistry: Concepts and Applications.

WB Saunders Company. Philadelphia.

Kusa M. 1966. Lactate Dehydrogenase Isozyme Patterns of the Stickleback, Pungitius pungitius*'.J.A.F:

vol 2

Mc Clatchey K. 2001. Clinical Laboratory medicine. 2nd

Edition. Lippincott Williams and Wilkins. UK

Mc Kenzie, Henderson. 1983. Electrophoresis of Lactate Dehydrogenase Isoenzymes. Clin. Chem. 29/1:

189-195

Pagana, Deska. 1998. Mosbys Manual of Diagnostic and Laboratory Tests. Mosby Inc. St Louis

Saito, . Oohashi. 1964. Clinical methods for the determination of isozymes. Clinic All-round : 13, 773-

779.

Wu A. 1998. Cardiac markers. Humana Press. USA

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Clinical Post Lab 2