Measurements of the activity of enzymes in plasma are of value in the diagnosis and management of a wide variety of diseases. Most enzymes measured in plasma are primarily intracellular, being released into the blood when there is damage to cell membranes, but many enzymes, for example renin, complement factors and coagulation factors, are actively secreted into the blood, where they fulfil their physiological functions.
Small amounts of intracellular enzymes are present in the blood as a result of normal cell turnover. When damage to cells occurs, increased amounts of enzymes will be released and their concentrations in the blood will rise. However, such increases are not always due to tissue damage. Other possible causes include:
· increased cell turnover
· cellular proliferation (e.g. neoplasia)
· increased enzyme synthesis (enzyme induction)
· obstruction to secretion
· decreased clearance.
Little is known about the mechanisms by which enzymes are removed from the circulation. Small molecules, such as amylase, are filtered by the glomeruli but most enzymes are probably removed by reticuloendothelial cells. Plasma amylase activity rises in acute renal failure but, in general, changes in clearance rates are not known to be important as causes of changes in plasma enzyme levels.
Plasma contains many functional enzymes, which a actively secreted into plasma. For example, enzymes blood coagulation. On the other hand, there are a few non functional enzymes in plasma, which are coming out from cells of various tissues due to normal wear and tear. Their normal levels in blood are very low; but are drastically increased during cell death (necrosis) or disease. Therefore assays of these enzymes are very useful in diagnosis diseases.
Enzyme assays usually depend on the measurement the catalytic activity of the enzyme, rather than the concentration of the enzyme protein itself. Since each enzyme molecule can catalyze the reaction of many molecules of substrate, measurement of activity provides great sensitivity. It is, however, important that the conditions of the assay are optimized and standardized to give reliable and reproducible results. Reference ranges for plasma enzymes are dependent on assay conditions, for example temperature, and may also be subject to physiological influences. It is thus important to be aware of both the reference range for the laboratory providing the assay and the physiological circumstances when interpreting the results of enzyme assays.
One international unit is the amount of enzyme that will convert one micromole of substrate per minute per litre of sample and is abbreviated as U/L. The SI Unit (System Internationale) expression is more scientific, where or Katal (catalytic activity) is defined as the number of mole of substrate transformed per second per litre of sample. Katal is abbreviated as kat or k (60 U = 1 μkat and 1 nk = 0.06 U).
Disadvantages of enzyme assays
A major disadvantage in the use of enzymes for the diagnosis of tissue damage is their lack of specificity to a particular tissue or cell type. Many enzymes are common to more than one tissue, with the result that an increase in the plasma activity of a particular enzyme could reflect damage to any one of these tissues. This problem may be obviated to some extent in two ways:
first, different tissues may contain (and thus release when they are damaged) two or more enzymes in different proportions; thus alanine and aspartate aminotransferases are both present in cardiac and skeletal muscle and hepatocytes, but there is only a very little alanine aminotransferase in either type of muscle;
second, some enzymes exist in different forms (isoforms), colloquially termed isoenzymes (although, strictly, the term 'isoenzyme' refers only to a genetically determined isoform). Individual isoforms are often characteristic of a particular tissue: although they may have similar catalytic activities, they often differ in some other measurable property, such as heat stability or sensitivity to inhibitors.
After a single insult to a tissue, the activity of intracellular enzymes in the plasma rises as they are released from the damaged cells, and then falls as the enzymes are cleared. It is thus important to consider the time at which the blood sample is taken in relation to the insult. If taken too soon, there may have been insufficient time for the enzyme to reach the blood- stream and if too late, it may have been completely cleared. As with all diagnostic techniques, data acquired from measurements of enzymes in plasma must always be assessed in the light of whatever clinical and other information is available, and their limitations borne in mind.
LACTATE DEHYDROGENASE (LDH) (LD)
The total LDH is generally tested by reaction of the serum sample with pyruvate and NADH2. LDH will convert pyruvate to lactate, and in turn NADH is use up by the reaction.
Normal value of LDH in serum is 100-200 U/L. Values the upper range are generally seen in children. Strenuous exercise will slightly increase the value. LDH level is 100 times more inside the RBC than in plasma, and therefore minor amount of hemolysis will result in a false-positive test.
LDH and Heart Attack
In myocardial infarction, total LDH activity is increased, while H4 iso-enzyme is increased 5-10 times more.
Differential diagnosis: Increase in total LDH level is seen in hemolytic anemias, hepatocellular damage, muscular dystrophy, carcinomas, leukemias, and any condition which causes necrosis of body cells. Since total LDH is increased in many conditions, the study of isozymes of LDH is of great importance.
Isoenzymes of LDH
LDH enzyme is a tetramer with four subunits. But the subunit may be either H (heart) or M (muscle) polypeptide chains. These two are the products of two different genes.
Although both of them have the same molecular weight (32 kD), there are minor amino acid variations. So five combinations of H and M chains are possible; H4, H3M, H2M2, M3H and M4 varieties, forming five iso-enzymes. All these five forms are seen in all persons. M4 form is seen in skeletal muscles; it is not inhibited by pyruvate. But H4 form is seen in heart and is inhibited by pyruvate. Normally LDH-2 (H3M1) concentration in blood is greater than LDH-1 (H4); but this pattern is reversed in myocardial infarction; this is called flipped pattern. The iso-enzymes are usual ly separated by cellulose acetate electrophoresis at pH 8.6. They are then identified by adding the reactants finally producing a colour reaction. . Lactate dehydrogenase isoenzymes (as percentage of total):
LDH1 14-26 %
LDH2 29-39 %
LDH3 20-26 %
LDH5 6-16 %
It is used for the reaction shown in Figure:
Creatine → Creatine phosphate
It was called as creatine phosphokinase in old literature.
Normal serum value for CK is 15-100 U/L for males and 10-80 U/L for females.
CK and Heart Attack CK value in serum is increased in myocardial infarction. The CK level starts to rise within three hours of infarction. Therefore, CK estimation is very useful to detect early cases, where ECG changes may be ambiguous. The CK level is not increased in hemolysis or in congestive cardiac failure; and therefore CK has an advantage over LDH.
CK and Muscle Diseases he level of CK in serum is very much elevated in muscular dystrophies (500 -1500 IU/L). The level is very high in the early phases of the disease. In such patients a fall in CK level is indicative of deteriorating condition, because by that time, all muscle mass is destroyed. In female carriers of this X-linked disease (genotypically heterozygous), CK is seen to be moderately raised. CK level is highly elevated in crush injury, fracture and acute cerebrovascular accidents. Estimation of total CK is employed in muscular dystrophies and MB iso-enzyme is estimated in myocardial infarction.
Iso-enzymes of CK CK is a dimer; each subunit has a molecular weight of 40,000. The subunits are called B for brain and M for muscle. They are products of loci in chromosomes 14 and 19 respectively. Therefore three iso-enzymes are seen in circulation. Normally CK2 is only 5% of the total activity. Even doubling the value in CK2 (MB) iso-enzyme may not be detected, if total value of CK alone is estimated. Hence the detection of MB-iso-enzyme is important in myocardial infarction. CK-MB < 6 % of total CK in normal conditions.
The above three iso-enzymes are cytosolic. A fourth variety, called CK-mt is located in mitochondria and constitutes about 15% of total CK activity. Its gene is located in chromosome 15. CK1 may be complexed with immunoglobulin; and then termed macroCK. CK1-lgG causes false-positive diagnosis of myocardial infarction because it has an electrophoretic mobility close to CK2.
For quantitating MB iso-enzyme, anti-MM antiserum is added to the patient's serum. This will precipitate MM iso-enzyme. The supernatant serum is used for the CK estimation. Here it is assumed that BB isoenzyme is negligible in quantity, which is correct if there is no brain disease. CK iso-enzymes can also be identified by electrophoresis.
AST present in cytosol and mitochondria
ALT located in cytosol of liver
In the liver, the concentration of ALT per unit weight of the tissue is more than AST.
These enzymes are more important in assessing and monitoring the degree of liver cell
inflammation and necrosis.
The highest activities of ALT are found in hepatocytes and muscle cells.
Again the hepatocytes have very high activity of ALT.
Therefore elevations in serum ALT are considered to be relatively specific for liver disease.
AST may be elevated in other forms of tissue damage, such as myocardial infarction,
muscle necrosis and renal disorders.
In liver disease, the ALT level is increased markedly compared to AST.
In acute viral hepatitis there is a 100-1000 times increase in both ALT and AST but ALT
level is increased more than that of AST
ASPARTATE AMINO TRANSFERASE (AST)
It is also called as serum glutamate-oxaloacetate transaminase (SGOT). AST needs pyridoxal phosphate as co-enzyme. AST is estimated by taking aspartate, α-ketoglutarate, pyridoxal phosphate (vitamin B6) and patient' serum as the source of AST. The oxaloacetate formed may be allowed to react with dinitrophenyl hydrazine to produce a colour which is estimated colorimetrically at 520 nm.
Normal serum level of AST is 8-40 U/L or (0,1-0,45 mmol/(hour´L))
It is significantly elevated in myocardial infarction. It if moderately elevated in liver diseases. However, a marked increase in AST may be seen in primary hepatoma. AST has two iso-enzymes; cytoplasmic and mitochondrial. In mile degree of tissue injury, cytoplasmic form is seen in serum. Mitochondrial type is seen in severe injury.
Marked increase (10 to 100 times the upper adult reference limit):
Circulatory failure with 'shock' and hypoxia:
Acute viral or toxic hepatitis.
Cirrhosis (may be normal, but may rise to twice the upper adult reference limit):
Infectious mononucleosis (due to liver involvement):
Cholestatic jaundice (up to 10 times the upper adult reference limit):
Malignant infiltration of the liver (may be normal, but may rise to twice the upper reference
Skeletal muscle disease:
After trauma or surgery (especially after cardiac surgery):
Severe haernolytic episodes (of erythrocyte origin).
ALANINE AMINO TRANSFERASE (ALT)
It is also called as serum glutamate-pyruvate transaminase (SGPT). ALT needs pyridoxal phosphate as co-enzyme.
Normal serum level of ALT is 5-30 U/L or (0,1-0,68 mmol/(hour´L))
Very high values (100 to 1000 U/L) are seen in acute hepatitis, either toxic or viral in origin. Both ALT and AST are increased in liver diseases, but ALT >AST. Moderate increase (25 to 100 U/L) may be seen in chronic liver disease such as cirrhosis, and malignancy in liver. A sudden fall in ALT level in cases of hepatitis is a very bad prognostic sign.
Ritis coefficient (AST/ALT) in normal conditions is 1,33±0,42.
Marked increase (10 to 100 times the upper limit of the adult reference range circulatory failure with 'shock' and hypoxia:
Acute viral or toxic hepatitis.
Cirrhosis (may be normal or up to twice the upper adult reference limit): infectious
mononucleosis (due to liver involvement):
Liver congestion secondary to congestive cardiac failure:
cholestatic jaundice (up to 10 times the upper reference limit in adults); surgery or
extensive trauma and skeletal muscle disease (much less affected than AST)
Alkaline phosphatase (ALP)
It is a non- specific enzyme which hydrolyses aliphatic, aromatic or heterocyclic compounds. The pH optimum for the enzyme reaction is between 9 and 10. It is prodused by osteoblasts of bone, and localized in cell memmbranes (ecto-enzyme).
Normal serum level of ALP is 40-125 U/L or 0,5-1,3 mmol/(hour´ L).
In children the upper level of normal value may be more, becouse of the increased osteoblastic activity. Mild increase is noticed during pregnancy, due to production of placental isoenzyme.
Moderate (2-3 times) increase in ALP level is seen in hepatic diseases such as hepatitis, alcoholic hepatosis or hepatocellular carcinoma. Very high levels of ALP (10-12 times of upper limit) may be noticed in extrahepatic obstructions or cholestasis. ALP is produced by epithelial cells of biliary canaliculi and obstruction of bile with consequent irritation of epithelial cells leads to secretion of ALP into serum.
Drastically high levels of ALP (10-25 times of upper limit) are also seen in bone diseases where osteoblastic activity is enhanced such as Paget's disease, rickets, osteomalacia, osteoblastoma, metastatic carcinoma of bone and hyperparathyroidism (Paget's disease or osteitis deformans was described in 1877 by Sir James Paget).
Iso-enzymes of Alkaline Phosphatase
1. α-1 ALP moves in α -1 position, it is synthesised by epithelial cells of biliary canaliculi. It is about 10% of total activity and is increased in obstructive jaundice and to some extent in metastatic carcinoma of liver.
-2 heat labile ALP is stable at
-2 heat stable ALP will not be destroyed at
ALP is of bone origin and elevated levels are seen in bone diseases. This is
the most heat labile (destroyed at
5. γ-ALP is inhibited by phenylalanine and originates from intestinal cells. It is increased in ulcerative colitis. About 10% of plasma ALP are of intestinal variety.
6. The leucocyte alkaline phosphatase (LAP) is significantly decreased in chronic myeloid leukemia. It is increased in lymphomas.
ALP has different isoforms. Although ALP is a monomer, depending on the number of sialic acid residues, the charged groups differ. Such different forms are detected in agar gel electrophoresis.
NUCLEOTIDE PHOSPHATASE (NTP)
It is also known as 5' nucleotidase. This enzyme hydrolyses 5' nucleotides to corresponding nucleosides at an optimum pH of 7.5. It is a marker enzyme for plasma membranes and is seen as an ecto-enzyme (enzyme present on the cell membrane).
Usually, AMP is used as substrate, which is hydrolysed to adenosine and inorganic phosphate. The latter reacts with ammonium molybdate to produce the yellow ammonium phosphomolybdate, which is estimated colorimetrically. However, ALP will also catalyse the same reaction. Serum samples contain both ALP and NTP. These are distinguished by Nickel ions which inhibit NTP but not ALP.
Normal NTP level in serum is 2-10 U/L. It is moderately increased in hepatitis and highly elevated in biliary obstruction. Unlike ALP, the level is unrelated with osteoblastic activity and therefore is unaffected by bone diseases.
GAMMA GLUTAMYL TRANSFERASE (GGT)
The old name was gamma glutamyl transpeptidase. It can transfer γ-glutamyl residues to substrate. In the body it is used in the synthesis of glutathione. GGT has 11 iso-enzymes. It is seen in liver, kidney, pancreas, intestinal cells and prostate gland.
Normal serum value of GGT is 6-45 U/L in male and 5-30 U/L in female. It is slightly higher in normal males, due to the presence of prostate gland. This value is moderately increased in infective hepatitis and prostate cancers. The GGT level is highly elevated in alcoholism, obstructive jaundice and neoplasm's of liver. GGT-2 is positive for 90% of hepatocellular carcinomas. It is not elevated in cardiac or skeletal diseases.
GGT is a microsomal enzyme. Its activity is induced by alcohol, phenobarbitone and rifampicin. GGT is clinically important because of its sensitivity to detect alcohol abuse. GGT is increased in alcoholics even when other liver function tests are within normal limits. GGT level is rapidly decreased within a few days when the person stops to take alcohol. Increase in GGT level is generally proportional to the amount of alcohol intake.
ACID PHOSPHATASE (ACP)
It hydrolyses phosphoric acid ester at pH between 4 and 6. Methods for assay are the same as described for ALP; but the pH of the medium is kept at 5 to 5.4.
Normal serum value for ACP is 2.5-12 U/L or 0,025-0,12 mmol/(hour´ L).
ACP is secreted by prostate cells, RBC, platelets and WBC. Isoenzymes of ACP are described. Erythrocyte ACP gene is located in chromosome 2; osteoclast ACP gene is on chromosome 19; lysosomal gene is on 11 and prostate ACP gene is on 13. The prostate iso-enzyme is inactivated by tartaric acid. Cupric ions inhibit erythrocyte ACP. Normal level of tartrate labile fraction of ACP is 1 U/L.
ACP total value is increased in prostate cancer and highly elevated in bone metastasis of prostate cancer. In these conditions, the tartrate labile iso-enzyme is elevated. This assay is very helpful in follow up of treatment of prostate cancers. ACP is therefore an important tumour marker.
Since blood cells contain excess quantity of ACP, must be taken to prevent hemolysis while taking blood from the patient. Prostate massage may also increase to value. So blood may be collected for ACP estimation before per rectal examination of patient. ACP is present in high concentration in semen, a finding which is used in forensic medicine in investigation of rape.
The main indications for estimation are to help diagnose prostatic carcinoma and to monitor its treatment. The estimation is gradually being replaced by the measurement of plasma prostate specific antigen (PSA) a protein derived from the prostate. This test is more specific and sensitive for diagnosis and monitoring treatment. However, it may be raised in similar circumstances to those affecting prostatic ACP and is more expensive to estimate. ACP is more
useful for monitoring the treatment of a known case of disseminated prostatic carcinoma than for making the diagnosis.
PROSTATE SPECIFIC ANTIGEN (PSA)
It is produced from the secretory
epithelium of prostal gland. It is normally secreted into seminal fluid, where
it is necessary for the liquefaction of seminal coagulum. It is a serine
protease, and is a 32 kD glycoprotein; encoded in chromosome number
Normal value is 1 -5 µg/L. It is very specific for prostate activity. Values between 4-10 µg/L is seen in benign prostate enlargement; but values above 10 µg/L is indicative of prostate cancer.
Acetyl cholinesterase or true ChE or Type 1 ChE can act mainly on acetyl choline. It is present in nerve endings and in RBCs. About 25 allelic forms are reported. Normal serum range is 2-12 U/ml. Newly formed RBC will contain good quantity of ChE which is slowly reduced according to the age of the cell. Therefore, ChE level in RBCs will be proportional to the reticulocyte count. Organophosphorus insecticides (Parathione) irreversibly inhibit ChE in RBCs. Measurement of ChE level in RBCs is useful to determine the amount of exposure in persons working with these insecticides.
Pseudocholinesterase or type II ChE is non-specific and can hydrolyse acyl esters. It is produced mainly by liver cells. Normal serum level is 8-18 U/ml. Succinyl choline is a widely used as muscle relaxant. It is a structural analogue of ACh, and so competitively fix on post-synaptic receptors of ACh. Succinyl choline is hydrolysed by the liver ChE within 2-4 minutes. But in certain persons the ChE activity may be absent; this is a genetically transmitted condition. In such individuals when succinyl choline is given during surgery, it may take hours to get the drug metabolised. Very prolonged scoline apnoea may result in 'nightmare of anaesthetist'. The pseudocholinesterase level in serum is reduced in viral hepatitis, cirrhosis, hepatocellular carcinoma, metastatic cancer of liver and in malnutrition.
Causes of decreased plasma cholinesterase activity
• Hepatic parenchymal disease (reduced synthesis).
• Ingestion or absorption through the skin, of such anticholinesterases as organophosphates.
• Inherited abnormal cholinesterase variants, with low biological activity.
Causes of increased plasma cholinesterase activity
a. Recovery from liver damage (actively growing hepatocytes)
b. Nephrotic syndrome
GPD is a dimer with identical subunits. This is an important enzyme in the hexose monophosphate shunt pathway of glucose. It is mainly used for production of NADPH . It has a special role in the RBC metabolism. Due to the presence of oxygen, hydrogen peroxide is continuously formed inside the RBC. Peroxide will destroy biomembranes, and RBCs are lysed. Normal value of GPD in RBC is 125-250 U/1012 cells. Nearly 400 variants (isoforms) of GPD are described.
This splits starch to maltose. It is activated by calcium, chloride and fluoride ions. There are 18 phenotypes. It is produced by pancreas and salivary glands; they are products of different genes located in chromosome 1.
Normal serum value is 50-120 U/L, (12-32 g/(hour× L)).
The value is increased about 1000
times in acute pancreatitis which is a life-threatening condition. The peak
values are seen between 5-12 hours after the onset of disease and returns to
normal levels within 2-4 days after the acute phase has subsided. Moderate
increase in serum levels are seen in chronic pancreatitis, mumps (parotitis),
obstruction of pancreatic duct and in renal disease. In the last condition, the
enzyme is not excreted through urine properly and hence serum value is raised. Normal
urine value is 20-160 g/(hour× L) or (less than 375 U/L). It is increased in acute pancreatitis. It is
increased on the
It will hydrolyse triglyceride to β-monoglyceride and fatty acid. Molecular weight is 54,000. The gene is in chromosome 10. The enzyme is present in pancreatic secretion. Normal serum range is 0.2-1.5 U/L. It is highly elevated in acute pancreatitis and this persists for 7-14 days. Thus, lipase remains elevated longer than amylase. Moreover, lipase is not increased in mumps. Therefore, lipase estimation has advantage over amylase. It is moderately increased in carcinoma of pancreas, biliary diseases and perforating peptic ulcers.
It is a tetrameric enzyme with A and B subunits; so there are 5 iso-enzymes. It is a glycolytic enzyme. Normal range of serum is 1.5-7 U/L. It is drastically elevated in muscle damages such as progressive muscular dystrophy, poliomyelitis, myasthenia gravis and multiple sclerosis. It is a very sensitive early index in muscle wasting diseases.
It is a glycolytic enzyme. Neuron-specific enolase (NSE) is an iso-enzyme seen in neural tissues and Apudomas. NSE is a tumour marker for cancers associated with neuro-endocrine origin, small cell lung cancer, neuroblastoma, pheochromocytoma, medullary carcinoma of thyroid, etc. It is measured by RIA or ELISA. Upper limit of NSE is 12 μg/ml.
Enzymes in Malignancy
Plasma total enzyme activities may be raised or an abnormal isoenzyme detected, in several neoplastic disorders.
• Serum prostatic (tartrate-labile) acid phosphatase activity rises in some cases of malignancy of the prostate gland.
• Any malignancy may be associated with a non-specific increase in plasma LD1 (HBD) and occasionally, transaminase activity.
• Plasma transaminase and alkaline phosphatase estimations may be of value to monitor treatment of malignant disease. Raised levels may indicate secondary deposits in liver or of alkaline phosphatase, in bone. Liver deposits may also cause an increase in plasma LD or GGT.
• Tumors occasionally produce a number of enzymes, such as the 'Regan' ALP isoenzyme.'
LD (HBD) or CK-BB. assays of which may be used as an aid to diagnosis or for monitoring treatment.
Other Clinical correlations
1. Niemann-Pick disease: Acid Sphingomyelinase Deficiency
· Sphingomyelin, a ubiquitous component of cell membranes, especially neuronal membranes, is normally degraded within lysosomes by the enzyme sphingomyelinase.
· In patients with Niemann-Pick disease, inherited deficiency of this enzyme causes spingomyelin to accumulate in lysosomes of the brain, bone marrow, and other organs.
· Enlargement of the lysosomes interferes with their normal function, leading to cell death and consequent neuropathy.
· Symptoms include failure to thrive and death in early childhood as well as learning disorders in those who survive the postnatal period.
2. Homocysteinuria: Cystathionine β-synthase Deficiency
1. Cystathionine β-synthase catalyzes conversion of homocysteine to cystathionine, a critical precursor of cysteine.
2. Deficiency of this enzyme leads to the most common form of homocystinuria, a pediatric disorder characterized by accumulation of homocysteine and reduced activity of several sulfotransferase reactions that require this compound or its derivatives as substrate.
3. Accumulation of homocysteine and reduced transsulfation of various compounds leads to abnormalities in connective tissue structures that cause altered blood vessel wall structure, loss of skeletal bone density (osteoporosis), dislocated optic lens (ectopia lentis), and increased risk of blood clots.
3. Enzyme Replacement Therapy for Inborn Errors of Metabolism
· Lysosomal enzyme deficiencies, which frequently result in disease due to accumulation of the substrate for the missing enzyme, are suitable targets for enzyme replacement therapy (ERT).
· In ERT, intravenously administered enzymes are taken up directly by the affected cells through a receptor-mediated mechanism.
· ERT provides temporary relief of symptoms but must be given repeatedly and is not a permanent cure.
1. Enzyme concentrations are high in cells. Natural decay of these cells releases enzymes into the plasma. Plasma activities are usually low but measurable.
2. Plasma enzyme assays are most useful in the detection of raised levels due to cell damage.
3. Assays of selected enzymes may help to identify the damaged tissues and isoenzyme studies may increase the specificity. In general, knowledge of the patterns of enzyme changes, together with the clinical and other findings, are needed if a useful interpretation is to be made.
4. Non-specific causes of raised enzyme activities include peripheral circulatory insufficiency, trauma, malignancy and surgery.
5. Artefactual increases may occur in haemolysed samples.
6. Enzyme estimations may be of value in the diagnosis and monitoring of:
a. • Myocardial infarction (CK. LD and its isoenzymes and sometimes AST);
b. • Liver disease (transaminases. ALP and sometimes GGT):
c. • Bone disease (ALP):
d. • Prosratic carcinoma (tartrate-labile ACP);
e. • Acute pancreatitis (α-amylase):
f. • Muscle disorders (CK)