Figure 1.

LDH isozyme banding patterns of heart, liver, muscle serum obtained from polyacrylamide gel electrophoresis (PAGE)

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

Figure 2 Results of polyacrylamide gel electrophoresis of serum proteins

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:

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).

(Gennady P. Manchenko. 2003) Five bands can be separated. The isoenzyme LDH1 is made up of four H subunits, it is found in the heart muscle and it is the most anodic of the five isoenzymes .Whilst the LDH5 isoenzyme is made up of four M subunits, this is located in skeletal muscle and also in the liver, this is the most cathodic of all the five isoenzymes. LDH2 LDH3 and LDH 4 are found in varying amounts in many tissues. Quantitation of these fractions provides useful information on the identification of specific tissue or organ damage. Electrophoresis of LDH isoenzymes is an important test for the diagnosis of myocardial and liver damages. (Wu 1998). Due to their different amino acid compositions the LDH isoenzymes can be separated by electrophoretic methods (in this case polyacrylamide is used as the matrix). In an electric field the LDH-1 migrates fastest towards the anode while the LDH-5 is the slowest isoenzyme. The mobility of a molecule in an electric field is inversely proportional to molecular friction which is the result of its molecular size and shape, and directly proportional to the voltage and the charge of the molecule. Proteins such as enzymes could be resolved electrophoretically in a semi-solid matrix strictly on the basis of molecular weight if, at a set voltage, a way could be found to charge these molecules to the same degree and to the same sign. Under these conditions, the mobility of the molecules would be simply inversely proportional to their size. (Manchenko 2003).

(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+ 2) NADH + PMS 3) PMS-H + TNBT Pyruvate + NADH + H+ NAD+ + PMS-H (PMS - Phenazine methosulfate)

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

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).

Sucrose was used when making up each solution, the reason for this its so that the solution will be 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,

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.

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 that LDH4 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

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).

(Bhagavan N. 1992.) Assaying LDH activity is based on the fact that NADH has an absorbance maximum around 340 nm which cannot be found in the spectra of NAD. So when the enzyme assay is carried out using oxo-substrate and NADH the time dependent decrease in the optical activities of the samples measured at 340 nm is proportional to the LDH activity (e340=6.2x106 M-1cm2). (Bhagavan 2002) A solution which contained lithium lactate, NAD, methyl phenazonium methosphosphate (PMS) and nitroblue tetrazolium was used for staining in this part of the experiment. The lactate was used in this staining method as a substrate for the LDH enzymes so that it could be oxidized by the enzymes, and give up an electron to the coenzyme and electron carrier NAD+. The NADH produced would then give up the electron to the PMS which was acting like an electron carrier, which would then give up the electron to the yellow tetrazolium salt, reducing the salt and producing a red color. The activity of the LDH isozymes on the lactate substrate would Therefore give rise to a color change in the tetrazolium salt, thus allowing the bands in the polyacrylamide gels to be identified. The bands which had a darker color would be interpreted to have a greater concentration of the LDH enzyme.( Bhagavan N. 1992.)

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)

Blue precipitate of formazan

(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 disease processes. In this type of electrophoresis, proteins are separated based on their relative mobilities in an electric field. (Mc Clatchey 2001).

(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)

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 disease in the patient, If a thick band was obtained lets say 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.

A source of error which occurred in this experiment was that there was overstraining. Overstaining is the retention of excess stain within the gel which results in a high background stain. The background stain prevents the protein bands from being seen. One way to prevent this from happening would be to ensure that the gel is not allowed to stain for too long or to simply closely monitor the gels as they are staining so that they are not left for too long leading to overstaining. Precautions : The samples were introduced into the wells slowly using a micropipette in order to avoid disturbing the molecular linking of the gels. The layer of butter was introduced over the sample in the wells very slowly in order to avoid disturbing the samples in the wells and to disrupt the gradual movement of the proteins through the gels.

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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, 773779.

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