Isoenzymes are enzymes that catalyze identical chemical reactions but are composed of different amino acid sequences. They are sometimes referred to as isozymes. Isoenzymes are produced by different genes and are not redundant despite their similar functions. They occur in many tissues throughout the body and are important for different developmental and metabolic processes.
Isoenzymes are useful biochemical markers and can be measured in the bloodstream to diagnose medical conditions. Isoenzymes can be differentiated from one another using gel electrophoresis. In gel electrophoresis, isoenzyme fragments are drawn through a thick gel by an electric charge. Each isoenzyme has a distinct charge of its own because of its unique amino acid sequence. This enables gel electrophoresis to separate the fragments into bands for identification. Some clinically important isoenzymes are as follows
1) Creatine Kinase(CK, CPK) is an enzyme found primarily in the heart and skeletal muscles, and to a lesser extent in the brain but not found at all in liver and kidney. Small amounts are also found in lungs, thyroid and adrenal glands. Significant injuryto any of these structures will lead to a measurable increase in CK levels. It is not found in red blood cells and its level is not affected by hemolysis.
Normal Value- serum activity varies from 10-50 IU/L at 30°C.
Elevations are found in:
After myocardial infarction- serum value is found to increase within hours, reaches a peak level in 24- 30 hours and returns to normal level in 2-4 days (usually in 72 hours). CK is a sensitive indicator in the early stages of myocardial ischemia. No increase in activity is found in heart failure and coronary insufficiency. In acute MI, CPK usually rises faster than SGOT and returns to normal faster than the SGOT.
CK/CPK Isoenzymes
There are three Isoenzymes. Measuring them is of value in the presence of elevated levels of CK or CPK to determine the source of the elevation. Each iso enzyme is a dimer composed of two protomers‘M’ (for muscles) and ‘B’( for Brain). These isoenzymes can be separated by, Electrophoresis or by Ion exchange Chromatography. The three possible iso enzymes are;
There are three Isoenzymes. Measuring them is of value in the presence of elevated levels of CK or CPK to determine the source of the elevation. Each iso enzyme is a dimer composed of two protomers‘M’ (for muscles) and ‘B’( for Brain). These isoenzymes can be separated by, Electrophoresis or by Ion exchange Chromatography. The three possible iso enzymes are;
| Isoenzyme | Electrophoretic mobility | Tissue of origin | Mean percentage in blood |
| MM(CK3) | Least | Skeletal muscle |
Heart muscle97-100%MB(CK2)IntermediateHeart muscle0-3% BB(CK1)MaximumBrain0%
o Macro CK(CK-Macro)- It is formed by the aggregation of CK-BB with immunoglobulins usually with IgG but sometimes Ig A.It may also be formed by complexing CK-MM with lipoproteins. No specific disease has been found to be associated with this isoenzyme.
o Mitochondrial CK(CK-Mi)- It is presentbound to the exterior surface of the inner mitochondrial membrane of muscle,liver and brain. It can exist in dimeric form or as oligomeric aggregates having molecular weight of approximately 35,000. It is only present in serum when there is extensive tissue damage causing breakdown of mitochondrial membrane and cell membrane. Thus its presence in serum indicates severe illness and cellular damage. It is not related with any specific disease states but it has been detected in certain cases of malignant tumors.
2) Aspartate amino Transferase (AST)
It is also called as Serum Glutamate Oxalo acetate Transaminase (SGOT). The level is significantly elevated in Acute MI.
Normal Value- 0-41 IU/L at 37°C
In acute MI- Serum activity rises sharply within the first 12 hours, with a peak level at 24 hours or over and returns to normal within 3-5 days. The rise depends on the extent of infarction. Re- infarction results in secondary rise of SGOT.
Prognostic significance- Levels> 350 IU/L are due to massive Infarction(Fatal),
> 150 IU/L are associated with high mortality and levels < 50IU/L are associated with low mortality.
Other diseases- The rise in activity is also observed in muscle and hepatic diseases. These can be well differentiated from simultaneous estimations of other enzyme activities like SGPT etc, which do not show and rise in activity in Acute MI.
3) Alanine amino transferase (ALT)- Also called serum Glutamate pyruvate transaminase.
Normal serum level ranges between 0-45 IU/L at37oC.
Very high values are seen in Acute hepatitis,toxic or viral in origin. Both ALT and AST rise but ALT> AST. Moderate increase may be seen in chronic liver diseases such as Cirrhosis and Malignancy in liver. A sudden fall in AST level in hepatitis signifies bad prognosis.
4) Lactate dehydrogenase (LDH)
Lactate dehydrogenase catalyzes the reversible conversion of pyruvate and lactate. LDH is essential for anaerobic respiration. When oxygen levels are low,LDH converts pyruvate to lactate, providing a source of muscular energy.
Normal level- 55-140IU/L at 30°C. The levels in the upper range are generally seen in children. LDH level is 100 times more inside the RBCs than in plasma, and therefore minor amount of hemolysis results in false positive result.
In Acute MI-The serum activity rises within 12 to 24 hours, attains a peak at 48 hours (2 to 4 days) reaching about 1000 IU/L and then returns gradually to normal from 8 th to 14 th day. The magnitude of rise is proportional to the extent of myocardial infarction. Serum LDH elevation may persist for more than a week after CPK and SGOT levels have returned to normal levels.
Other diseases-The increase in serum activity of LDH is also seen in hemolytic anemia, hepatocellular damage, muscular dystrophies,carcinoma, leukemias, and any condition which causes necrosis of the body cells. Since the total LDH is increased in many diseases, so the study of Isoenzymes of LDH is of more significance.
Iso enzymes of LDH
LDH enzyme is tetramer with 4 subunits. The subunit may be either H(Heart) or M(Muscle) polypeptide chains. These two chains are the product of 2 different genes. Although both of them have the same molecular weight, there are minor amino acid variations.There can be 5 possible combinations; H4, H3M1, H2M2, H1M3. M4, these are 5different types of isoenzymes seen in all individuals.
| No. of Isoenzyme | Subunit make up of isoenzyme | Electrophoretic mobility at pH8.6 | Activity at 60°for 30 minutes | Tissue origin | Percentage in human serum(Mean) |
| LDH-1 | H4 | Fastest | Not destroyed | Heart muscle | 30% |
| LDH-2 | H3M1 | Faster | Not destroyed. | RBC | 35% |
| LDH-3 | H2M2 | Fast | Partly destroyed | Brain | 20% |
| LDH-4 | H1M3 | Slow | Destroyed | Liver | 10% |
| LDH-5 | M4 | Slowest | Destroyed | Skeletal Muscles | 5% |
Normally LDH- 2(H3M1) level in blood is greater than LDH-1, but this pattern is reversed in myocardial infarction, this is called ‘flipped pattern’. These iso enzymes are separated by cellulose acetate electrophoresis at pH 8.6.
5) Alkalinephosphatase (ALP )-is an enzyme that removes phosphate groups from organic or inorganic compounds in the body. It is present in a number of tissues including liver, bone, intestine,and placenta. The activity of ALP found in serum is a composite of isoenzymes from those sites and, in some circumstances, placental or Regan isoenzymes. The optimum pH for enzyme action varies between 9-10. It is a zinc containing metalloenzyme and is localized in the cell membranes (ectoenzyme). It is associated with transport mechanism in the liver, kidney and intestinal mucosa.
Normal serum Level- of ALP ranges between 40-125 IU/L. In children the upper level of normal value may be more, because of increased osteoblastic activity.
Total Alkaline Phosphatase (ALP)
Serum ALP is of interest in the diagnosis of 2 main groups of conditions-hepatobiliary diseases and bone diseases associated with increased osteoblastic activity.
Mild increase is observed in pregnancy, due to production of placental enzyme.
A moderate rise in ALP activity occurs inhepatic diseases such as infective hepatitis, alcoholic hepatitis or hepatocellular carcinoma. Moderate elevation of ALP may also be seen in other disorders such as Hodgkin’s disease, congestive heart failure, ulcerative colitis, regional enteritis, and intra-abdominal bacterial infections.
ALP elevations tend to be more marked (more than 3-fold) in extra hepatic biliary obstructions (eg, by stone or cancer of the head of the pancreas) than in intrahepatic obstructions, and the more complete the obstruction, the greater the elevation. With obstruction, serumALP activities may reach 10 to 12 times the upper limit of normal, returning to normal upon surgical removal of the obstruction. The ALP response to cholestatic liver disease is similar to the response o fgamma-glutamyltransferase (GGT), but more blunted. If both GGT and ALP are elevated, a liver source of the ALP is likely.
The response of the liver to any form of biliary tree obstruction is to synthesize more ALP. The main site of new enzyme synthesis is the hepatocytes adjacent to the biliary canaliculi.
The response of the liver to any form of biliary tree obstruction is to synthesize more ALP. The main site of new enzyme synthesis is the hepatocytes adjacent to the biliary canaliculi.
ALP also is elevated in disorders of the skeletal system that involve osteoblast hyperactivity and bone remodeling, suchas Paget’s disease rickets and osteomalacia, fractures, and malignant tumors.
Among bone diseases, the highest level o f ALP activity is encountered in Paget’s disease, as a result of the action of the osteoblastic cells as they try to rebuild bone that is being resorbed by the uncontrolled activity of osteoclasts. Values from 10 to 25 times the upper limit of normal are not unusual.
Only moderate rises are observed in osteomalacia, while levels are generally normal in osteoporosis. In rickets,levels 2 to 4 times normal may be observed. Primary and secondary hyperparathyroidism are associated with slight to moderate elevations of ALP;the existence and degree of elevation reflects the presence and extent of skeletal involvement.
Very high enzyme levels are present in patients with osteogenic bone cancer.
Very high enzyme levels are present in patients with osteogenic bone cancer.
A considerable rise in ALP is seen in children following accelerated bone growth.
Patients over age 60 can have a mildly elevated alkaline phosphatase (1–1½ times normal), while individuals with blood types O and B can have an elevation of the serum alkaline phosphatase after eating a fatty meal due to the influx of intestinal alkaline phosphatase into the blood.
ALP Isoenzymes
Electrophoresis is considered the most useful single technique for ALP isoenzyme analysis. By starch gel electrophoresis at pH 8.6 , at least 6 isoenzyme bands can be visualized.
1) Hepatic ALP isoenzyme- moves fastest towards the anode and occupies the same position as Alpha 2 globulins. It is associated with biliary epithelium and is elevated in cholestatic processes. Various liver diseases (primary or secondary cancer, biliary obstruction) increase the liver isoenzyme.
2) Bone isoenzyme- It closely follows the hepatic enzyme and occupies the beta region. Osteoblastic bone tumors and hyperactivity of osteoblasts involved in bone remodeling (eg, Paget’s disease)increase the bone isoenzyme. Paget’s disease leads to a striking, solitary elevation of bone ALP.
3) Placental isoenzyme follows bone isoenzyme. It is heat stable isoenzyme and increases during last six weeks of pregnancy.
4) The intestinal isoenzyme-is the slowly moving band and follows the placental isoenzyme. It may be increased in patients with cirrhosis and in individuals who are blood group O or B secretors. Increased levels are also sen in patients undergoing hemodialysis.
Atypical ALP isoenzymes (Oncogenic markers)-In addition to 4 major isoenzymes, 2 more abnormal fractions are seen associated with tumors. These are Regan and Nagao isoenzymes. They are also called “Carcino placental ALP”, as they resemble placental isoenzymes.
Regan isoenzyme is elevated in various carcinomas of breast, lungs, colon and ovaries. Highest positivity is observed in carcinoma of ovary and uterus.
Atypical ALP isoenzymes (Oncogenic markers)-In addition to 4 major isoenzymes, 2 more abnormal fractions are seen associated with tumors. These are Regan and Nagao isoenzymes. They are also called “Carcino placental ALP”, as they resemble placental isoenzymes.
Regan isoenzyme is elevated in various carcinomas of breast, lungs, colon and ovaries. Highest positivity is observed in carcinoma of ovary and uterus.
Rise in Nagao isoenzyme is observed in metastatic carcinoma of pleural surfaces and adenocarcinoma of pancreas and bile duct.
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