PLASMA ENZYMES An enzyme is a protein with specific catalytic function.

Its presence in small quantities results in a large increase in the rate of substrate utilization and product formation in vitro. As such it is easier to measure enzyme activity in body fluids than its concentration which is normally low. Many enzymes are present in plasma of healthy individuals and more are found in disease conditions. There are two main categories of plasma enzymes: 1. Functional Enzymes (plasma-active) These are enzymes with a clearly defined function in blood. E.g. coagulation factors such as prothrombin factor V, VII and XI and also lipoprotein lipase. These enzymes originate from the liver and their concentration in blood is higher than in other tissues. A reduction in the synthesizing capacity of the organ of origin which may be due to decrease in the number of intact cells and/or to cellular damage in the organ of origin, will cause a depression of the enzymes activities in the plasma. 2. Non-Functional Enzymes. These are enzymes with no known function in the blood and which have been released from cells as a result of leakage or of cell death in the normal process of wear and tear of tissues, or due to diffusion through undamaged cell membranes. It is the activity measurements of the non-functional enzymes which help in the resolution of the problems of diagnosis. Generally the level of plasma enzymes is maintained in a steady state because their rate of diffusion into the plasma is balanced by the rate of disposal. Alterations in the activity of plasma enzymes are observed in the event of the following: a) b) c) d) e) Altered synthesis of the enzymes within the cells. Change in the amount of enzyme forming tissue. Change in cell permeability. Change in the rate of inactivation or disposal of enzyme. Obstruction to a normal pathway of enzyme excretion.

The rate and extent of enzyme release may be affected by the following factors: a) The amount of the enzyme within the cell. b) The molecular weight of the enzyme; small molecules are released more readily than large ones. c) The intracellular site of the enzyme; cytoplasmic enzymes are released more readily than mitochondrial enzymes. Enzyme assays provide useful clinical information by: a. Identifying the presence of disease. b. Distinguishing the specific disease from the other diseases. c. Providing estimates of the severity and duration of disease. Serum is preferred to plasma in enzyme assays since anti-coagulation factors may have inhibitory effects on the activity of the enzyme.
Lecture notes on Plasma Enzymes. 2007

CONSIDERATIONS IN THE SELECTION OF ENZYME ASSAYS In organ damage, increased amounts of many enzymes are released into the blood but only few of these are selected for diagnosis. The choice of enzymes and technique employed depends on the following factors: 1. Sensitivity The technique must be capable of detecting small amounts of an enzyme by its activity measurement. This helps overcome the problem of making a clinical diagnosis when tissue damage is minimal. 2. Specificity This is the ability to identify which tissue has been damaged. This problem may be overcome by measuring several different enzyme activities or by the study of is enzymes. 3. The Rate of Release Usually increased enzyme activity in plasma occurs three hours after tissue damage. Depending on the pathology of the condition its activity rises more or less quickly to its maximum. 4. Duration of Release The activity of the enzyme should be elevated soon after the onset of the disease and should persist appreciable period. This depends solely on the nature and extent of pathological process. 5. Half-Life of the Enzyme Some enzymes have very short half-lifes whereas others have long lifes i.e. 5 days or more. 6. Technical Factors The test must be precise and accurate, easy to perform and inexpensive. ISOENZYMES When multiple forms of an enzyme which catalyze the same reaction occur in the same species, especially in one tissue of an individual animal, the different forms are referred to as Isoenzymes. Isoenzymes may originate from different organs (e.g. alkaline phosphatase) or from different cell compartments (e.g. AST) or from the same compartment (e.g. LDH). The isoenzymes can be distinguished by their biochemical and physical properties. Isoenzymes can be differentiated by their distinctive behavior during electrophoresis (i.e. surface charge) and their different reactions to heat, inhibitors, activators, ion-exchange chromatography, isoelectric focusing and sometimes antisera. The most popular of these detection methods has undoubtedly been electrophoresis (using cellulose acetate, agar, and starch or acrylamide gel). Sometimes it is possible to detect differences between isoenzymes by using a different substrate. They may be distinguished by their kinetic properties, i.e. Michaelis constant (Km and Vmax) or by different sensitivities to temperature changes. ENZYMES OF CLINICAL INTEREST 1. LACTATE DEHYDROGENASE (LDH) This enzyme catalyzes the reversible interconversion of lactate and pyruvate: Lactate + NAD+ Pyruvate + NADH + H+
Lecture notes on Plasma Enzymes. 2007

It is widely distributed in the body with high concentrations in heart and skeletal muscle, liver, kidney, brain and erythrocytes. LDH (molecular weight of about 135,000) exists in body tissues as a tetramer consisting of four polypeptide chains each with a molecular weight of 33,000. Each chain is formed from one of two distinct monomers; H and M which can combine in various proportions to give rise to five isomers according to the chain structure as H4, H3M, H2M2, HM3 and M4. H4 is predominant in the heart muscle whereas M4 is predominant in the skeletal muscle. H4 has a high Michaelis constant (Km) for pyruvate. Its activity is inhibited by a high pyruvate concentration. M4 on the other hand has a low Km for pyruvate reduction to lactate. An additional minor LDH is present in the testis. The isoenzymes may be classified into LD1 to LD5 depending on the speed of migration towards the anode during electrophoresis at pH of 7.4 (physiological pH). LD1 migrates most rapidly towards the anode whereas LD5 is the slowest. It is possible to divide most tissues into one of the three categories. 1) Tissues in which the fast moving LD1 and LD2 (anodal isoenzymes) predominate e.g. heart, red blood cells. 2) Tissues in which the slow moving LD5 and LD4 (cathodal isoenzymes) predominate e.g. liver and most skeletal muscles. 3) Tissues in which no single component predominates e.g. lung and spleen. Elevation of LD1 and LD2 (LD1 greater than LD2) occurs in myocardial infarction, megaloblastic anaemia and renal infarction. Elevation of LD2 and LD3 occurs in acute leukemia. LD3 is the main isoenzyme elevated in many other malignancies. Elevation of LD5 occurs after damage to liver or skeletal muscle. The various isoenzymes may be distinguished from each other by the fact that LD1 and to a small extent LD2 and LD3 can utilize -hydroxybutyrate as well as lactate as substrate whereas LD4 and LD5 cannot. CH3CH(OH)COOH Lactate CH3CH(OH)CH2COOH -hydroxybutyrate

The normal level for serum lactate dehydrogenase is 130-500 U/l at 37oC in adults. Elevations more than five times normal may be observed in myocardial infarction and some haematological disorders. In pernicious anaemia and leukemia elevations up to twenty normal may be found. Lesser increases occur in other states of abnormal erythropoiesis e.g. thalasemia, myelofibrosis and haemolytic anaemia. Moderate increases are observed in the following conditions: viral hepatitis, malignancies, skeletal muscle disease, pulmonary embolism, and infectious mononucleosis.

2. CREATINE KINASE (CK) It is also incorrectly referred to as creatine phosphokinase. CK catalyzes the reversible phosphorylation of creatine by adenosine triphosphate (ATP).
Lecture notes on Plasma Enzymes. 2007

pH 9.0 Mg2+

Creatine + ATP

pH 6.7

Phosphocreatine + ADP

CK activity is greatest in striated muscle, brain and heart tissues. Less activity may be found in tissues like smooth muscle, kidney and the diaphragm. CK is a dimer formed from different combinations of two types of polypeptide chains or subunits designated as B or brain and M or muscle. Three different pairs of subunits and therefore three principal isoenzymes forms exist: BB (CK-1), MB (CK-2) and MM (CK-3). They have been numbered CK1, CK2 and CK3 on the basis of their electrophoretic mobility. The MM isoenzyme (CK-MM) predominates in the skeletal and cardiac muscles; the MB isoenzyme (CK-MB) is found in heart muscle. Its presence in plasma suggests a myocardial infarction. The BB isoenzyme is also found in the brain, thyroid and the smooth muscle of the gastro-intestinal and genital tracts. All of them are found in the cell cytosol or are associated with myofibrillar structures. However, a 4th one exists; which differs from the others both immunologically and electrophorecticic mobility is designated as CK-Mt. This is located between the inner and outer membranes of mitochondria, and in the heart constitutes up to 15% of the total CK activity. CK activity may also be found in two macromolecular forms: macro CK types 1 and 2. Type 1 is CK-1 associated with IgG or CK-3 with IgA and type 2 is oligomeric CK-Mt. The normal range is : Female Male

3-70 U/L 5-100 U/L at 37oC

Plasma normally contains the MM isoenzyme and increase in total creatine kinase is always due mainly to MM isoenzyme. Marked increases of the enzyme are observed in the following: myocardial infarction, muscular dystrophies and Rhabdomyolosis (breakdown of skeletal muscle), shock and circulatory failure. Moderate increases are observed in the following: muscle injury, after surgery (for about a week), hypothyroidism, alcoholism and acute psychotic episodes. 3. ASPARTATE TRANSAMINASE (AST)/ GLUTAMATE OXALOACETATE TRANSAMINASE (GOT) This enzyme catalyzes the transfer of an amino group from glutamic acid to oxaloacetic acid to form ketoglutaric acid and aspartic acid respectively. Glutamic acid + Oxaloacetic acid -ketoglutaric acid + Aspartic acid

Though present in tissue in most tissues, it has a high activity in cardiac muscle, skeletal muscle, liver, kidney and erythrocytes. There are two major isoenzymes; one cytoplasmic and the other mitochondrial. The normal level is 5-42 U/L at 37% in adults.

Lecture notes on Plasma Enzymes. 2007

Marked elevations are observed in the following: myocardial infarction, viral hepatitis, toxic liver necrosis (localized death of tissue), circulatory failure with shock and hypoxia (diminished amount of oxygen).

Moderate elevations may be observed in the following: Cirrhosis (2X normal), Cholestatic jaundice or obstructive jaundice (10X normal)}, Malignant infiltration of the liver, Skeletal muscle disease, Severe haemolytic anaemia, Infectious mononucleosis:-increase in the number of monocytes or glandular fever:- most probably caused by Epstein-Barr virus causing an increase in circulating monocytes, After trauma (bodily injury, emotional shock) or surgery. 4. ALANINE TRANSAMINASE (ALT)/ GLUTAMATE PYRUVATE TRANSAMINASE (GPT) This enzyme catalyzes the transfer of an amino group from glutamic acid to pyruvic acid to form ketoglutaric acid and alanine respectively. Glutamic acid + Pyruvic acid -ketoglutaric acid + Alanine

Though widely distributed it is present in high concentrations in the liver and to a lesser extent in skeletal muscle, kidney and heart. The activity of ALT and AST is the same magnitude in the liver thus most laboratories employ only AST assays in their diagnosis. Marked elevations are observed in: Viral hepatitis, Toxic liver necrosis, Hypotension leading to circulatory failure with shock and hypoxia. Moderate elevations are observed in: Cirrhosis (2X normal), Cholestatic jaundice (10X normal), Liver congestion secondary to cardiac failure Infectious mononucleosis with liver involvement Extensive trauma and muscle disease (less than AST). An ALT/AST greater than 2 favors a diagnosis of alcohol induced hepatitis or cirrhosis. 5. ALDOLASE This enzyme catalyzes the splitting of fructose-1,6-diphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphte. The enzyme is widely distributed. It is present in the liver, muscle, erythrocytes and kidney. The activity of the enzyme is increased in patients with muscle diseases such as muscular dystrophy, and in patients with myocardial infarction, malignant disease, hepatocellulary damage and haemolytic anaemia. Since the advantages of aldolase over the enzyme assays such as AST, ALT and CK are few, if any; its activity is often not used in diagnoses.

Lecture notes on Plasma Enzymes. 2007

6. PSEUDOCHOLINESTERASE This is the plasma analogue of acetylcholinesterase, an enzyme which hydrolyzes acetylcholine. This enzyme which is synthesized chiefly in the liver is responsible for the hydrolysis of succinylcholine, an analogue of acetylcholine, which is used to produce muscular relaxation in surgical procedures. The reference range of pseudocholinesterase is 2-5 U/L at 37oC. Pseudocholinesterase levels are increased in the following: Recovery from liver damage Nephrotic syndrome Pseudocholinesterase activity is decreased in the following: Hepatitis Cirrhosis Anticholinesterases Inherited abnormal cholinesterase Myocardial infarction 7. ALKALINE PHOSPHATASE (ALP) Alkaline phosphate is a hydrolytic enzyme which derives its name from that its optimum activity occurs at the alkaline pH of 9-10.5. It is widely distributed enzyme which may be found in bone, liver, kidney, intestinal wall, lactating mammary and placenta (during pregnancy). In bone, the enzyme is found in osteoblasts where it has been proposed to be involved in bone deposition. Generally the enzyme is localized at the membrane border of cells and appears to be significant in transport processes involving phosphate. After a meal containing fats and oils, there may be a small increase in total alkaline phosphate activity due to the release of the intestinal isoenzyme into the plasma. This has led to the suggestion that the enzyme may be involved in lipid transport. As expected from its presence in osteoblasts, ALP and bone growth show a parallel association. Levels in infants rise rapidly within a few days after birth and 2-3 times the adult value from age 1 year through puberty. Additional increases occur during puberty, after which values fall to adult range. Because of its presence in the placenta, ALP is elevated during pregnancy, especially in the last trimester. Typical reference values for the various stages of life are: Children (1-12 years): up to 350 U/L During puberty: up to 500 U/L Adults (up to 60 years) 20-90 U/L Serum ALP determinations are of particular interest in the investigation of hepatobiliary and bone disease associated with increased osteoblastic activity. Hepatobiliary disease which characterized by an elevation serum ALP activity includes extra- and intra-hepatic cholestasis and infectious hepatitis. Among the bone disease which increases the activity of serum ALP are Pagets disease, primary and secondary hyperparathyroidism (moderate), osteomalacia, rickets and osteogenic bone cancer.

Lecture notes on Plasma Enzymes. 2007

Certain drugs are associated with increases in serum ALP. These include anabolic steroids, androgens, testosterone and antibiotics such as erythromycin, sulphonamides and tetracycline. Others are allopurinol, aspirin, coumarin, methyldopa and vitamin D. Low serum ALP activities are associated with hypophosphatasia, achondroplasia, cretinism and vitamin C deficiency. Low activities may be caused by chemicals such as arsenicals, beryllium, cyanide and high levels of lithium, potassium and zinc. Achondroplasia: inherited condition characterized by arrested growth of the long bone resulting in dwarfism. Pagets disease: excessive ALP causes too rapid bone formation; consequently bone is thin. There is loss of stature, crippling deformity, abnormally enlarged head, collapse of vertebrae; and nervous complication can result. Osteomalacia: infectious mineralization of the mature skeleton with softening and bone pain. It is commonly caused by insufficient dietary intake of vitamin D. Osteomyelitis: inflammation commencing in the marrow of bone. Rickets: a disorder of calcium and phosphorus metabolism associated with a deficiency of vitamin D; and beginning most often in infancy and early childhood between the ages of 6 months and 2 years. ENZYME PATTERNS IN DISEASE 1. MYOCARDIAL INFARCTION The most useful enzyme tests in myocardial infarction are AST, LDH and CK. In the investigation of this disorder, it is customary to measure activities of 2 or even all three of these enzymes as well as the isoenzymes of CK and LDH. The choice of estimation, however, depends on the time interval after suspected infarction. After a myocardial infarction there is an initial period during which all serum enzyme activities remain within their reference ranges. Thereafter, activities rapidly rise to peaks whose magnitudes are proportional to the extent of myocardial damage. The first enzyme to rise is the MM or CK-3 isoenzyme. The increase is equivalent to the total CK activity, which exceeds upper limit of the reference range in 5-12 hours after the onset of chest pain, reaches a peak within 18-30 hours and returns tom within the normal range within 2-5 days after the onset of the infarction. CK-2 or MB isoenzyme has a shorter half-life and returns to the normal range by or before the 3rd day. In fact the presence of CK-2 in plasma almost always suggests a myocardial infarction. Serum CK-2 or MB activity is recorded as a percentage of the total CK, the normal value being 6% of the total, but following an infarction values can increase to 30% depending on the size of the infarction, methods used for the analysis and the location of the infarct. Owing to the fact that the activity of CK can be increased for so many reasons including muscular exercise and intramuscular injections; AST and LDH, particularly the heart specific LDH (LDH1) may be employed in the diagnosis of this condition. However, given the lack of tissue specificity of AST, the most useful enzymes for diagnostic purposes in a patient with suspected myocardial infarction are CK

Lecture notes on Plasma Enzymes. 2007

for early confirmation of the diagnosis and LDH1 for those patients (often with atypical clinical features who may present several days after the supposed infarct). AST activity rises within 6-12 hours after the onset of chest pain, increases to a peak within 20-30 hours and returns to the pre-infarction levels by the 2nd to the 6th day. However, because AST is not cardiac specific it has a poor diagnostic performance. The heart specific LDH (LDH1) rises 8-16 hours after the onset of chest pains. The isoenzyme reaches a peak within 30-48 hours and returns to the normal range after 5-14 days. Owing to its long half-life, LDH1 is a very good marker of myocardial infarction. Diagnostic sensivity of LDH1 is 90%, diagnostic specificity is 90-99%. The Pattern of Plasma Enzyme Activity after the Onset of an Infarction ENZYME ONSET (HRS) PEAKELEVATION (HRS) DURATION (DAYS) CK-2 (MB) 3-10 12-24 1.5-3 CK (Total) 5-12 18-30 2-5 AST 6-12 20-30 2-6 Heart Specific LDH 8-16 30-48 5-14 A second rise in enzyme levels after their return to normal indicates an extension of the infarction or the development of congestive cardiac failure, in which case, LDH1 and CK levels do not increase. 2. LIVER DISEASE ALT, AST, ALP and -glutamyl transferases (-GT or GGT) are the enzymes most often employed in the diagnosis of liver disease. The degree of elevation of these cytosolic, mitochondrial and membraneassociated enzymes varies with the type of liver disease. It has been demonstrated that increases in cytosolic isoenzymes is due to the impairment of cellular energy production (as in viral hepatitis) increases the permeability of the hepatocytes membrane and allows cytosolic isoenzymes of the aminotransferases to spill into the sinusoids and thence into the peripheral blood. When permeability of mitochondrial membranes increases, mitochondrial enzymes are also released. Increase in the activities of membrane-bound enzymes such as -GT and ALP are believed to be due to enhanced synthesis of these enzymes in disease liver. Chronic impairment of hepatocellulary function is attributed to impaired synthesis of enzymes such as cholinesterase. Thus measurements of the different types of enzymes may be of value in differentiating the various types of jaundice. They are very useful in the detection of liver disease in the unjaundiced patient. Hemolytic Jaundice The activities of plasma enzymes are usually normal but heart specific LDH (LDH1) may be elevated owing to its release from the erythrocytes. NB: Jaundice: -icterus No jaundice: - anicterus Before jaundice: - preicterus Hepatocellular Disease
Lecture notes on Plasma Enzymes. 2007

ALT levels higher than AST may be observed in conditions such as reversible alcoholic hepatitis, chronic persistent hepatitis, or in early chronic active hepatitis. ALT is more liver specific than AST. An AST level higher than that of ALT may be due to cirrhosis or severe chronic active hepatitis. A high ALP suggests cholestasis. In order to distinguish between elevations of ALP activity due to hepatic or bone disease, it has been proposed that GGT estimations will be more sensitive index of cholestasis. However, the clinical application of GGT activity measurements has not yet been evaluated. Nevertheless, GGT activity is elevated in any and all forms of liver disease. It is highest in cases of intra- or post-hepatic biliary obstruction reaching some 5-30 times normal. It is more sensitive than ALP, 5 nucleotidase (5N) and the transaminases in detecting obstructive jaundice, cholangitis (inflammation of the bile duct), and cholecystitis (inflammation of the gall bladder). Its rise occurs earlier than with these other enzymes and persists longer. Only moderate elevations (2-5 times) are seen in infectious hepatitis, and in this condition, GGT determinations are less useful diagnostically than are measurements of the transaminases. 3. MUSCLE DISEASE The most valuable enzyme activities used for the diagnosis of muscle disease are CK and the transaminases. CK is the enzyme of choice in most laboratories because of its markedly raised values irrespective of the aetiology of the disorder. In all types of muscular dystrophy, and especially in the Duchene type (in which levels up to 50 times the upper limit is seen), serum CK activity is extremely raised at some time during the course of the disease. In progressive muscular dystrophy (especially Duchenes dystrophy which is usually transmitted as a sex-linked recessive disorder), the enzyme activity is greatest during infancy and in childhood (710years of age) and may rise long before the disease is clinically apparent. But as patient gets older and most of the muscle has wasted the level of activity falls and may even be normal. Serum CK activity is however normal in neurogenic muscle diseases such as myastemia gravis (muscular weakness characterized by marked fatiguability of voluntary muscles, especially those of the eye), multiple sclerosis (variable progressive disease of the nervous system commonly affecting young adults), poliomyelitis and parkinsonism. 4. BONE DISEASE Enzyme assays which are employed in the diagnosis of bone diseases ALP and -GT. The assay of -GT is to help in the differentiation between liver and bone disease. In bone disease, the activity of -GT is normal whereas that of ALP is elevated. In liver and biliary tree disease, however, both ALP and -GT are elevated. ALP is secreted by osteoblasts and an increased serum concentration is seen in many diseases of the bone. Osteoporosis is an important exception to this. Unless there is coexisting osteomalacia or a fracture occurs the ALP is normal. In multiple myeloma, despite extensive tumour deposition in the bone, the ALP is not raised since there is no concomitant increase in osteoblastic activity. The most marked elevation is observed in Pagets disease. Other diseases which are characterized by raised ALP activity are osteomalacia, rickets, primary hyperparathyroidism with bone disease,
Lecture notes on Plasma Enzymes. 2007

secondary carcinoma in bone and in some cases of osteogenic sarcoma. Serial measurements of serum ALP are of particular value in the assessment of healing in osteomalacia, rickets, renal osteodystrophy and in Pagets disease. In the event of conditions which are characterized by arrested bone growth such as Achondroplasia, cretinism, vitamin C deficiency and hypophosphatasia, the activity is very low. 5. METASTATIC CARCINOMA OF THE PROSTATE A. Acid Phosphatase (ACP) This is the generic name for a group of enzymes which hydrolyze phosphates at a pH of 5-6. It is abundant in prostate and seminal fluid, while small concentrations of other isoenzymes are present in liver, spleen, red cells, platelets, kidney and bone. The isoenzymes may be distinguished by measuring activity in the presence of selective inhibitors or by using a substrate that is preferentially hydrolyzed by one of the isoenzymes. Most laboratories report either tartrate labile or formaldehyde-stable acid phosphatase activity in addition to total plasma ACP activity. Degree of Inhibition Source of ACP L (+) tartrate Formaldehyde Prostate Almost complete Minimal Red cells Minimal Marked Other tissues (e.g. liver, spleen) Marked Moderate The reference range for total acid phosphatase in adult plasma is 0.5-5.5 U/L (0.3-3.0 King Armstrong units/dl). The reference value for plasma prostatic ACP is < 4.0 U/L. The determination of plasma ACP activity is of significance in the demonstration of the presence and extent of invasive or metastatic prostatic carcinoma, and the response of prostatic carcinoma to oestrogen therapy. In prostatic carcinoma, particularly if it has metastasized, plasma ACP levels rise probably because of increased numbers of prostatic cells. In cases when there are metastatic deposits in the bone (i.e. when these metastases are osteoblastic) plasma ACP level rises to 9 U/L. In cases of severe spread levels above 200U/L may be attained. In cases where the carcinoma is still confined within the capsule of the prostate, the Plasma Tartrate Labile ACP (TLACP) activity does not rise above the upper limit of reference values, as a general rule. If in a patient of prostatic carcinoma on oestrogen (e.g. stilbesterol) therapy, an early and often dramatic fall in TLACP activity in plasma is attained, it is an indication of response to treatment. Any subsequent elevation in plasma TLACP activity is an indication of escape from the control exerted by oestrogen therapy to treatment. It must be noted that increased plasma TLACP activity is not absolutely reliable as an indication of the presence of prostatic carcinoma. The prostatic isoenzyme of ACP is occasionally increased in plasma from patients within benign prostatic hyperplasia or with prostatic infection. In 75% or more of cases with metastasing prostatic carcinoma however, there are marked elevations. The following precautions must be observed during ACP determinations for suspected cases of prostatic carcinoma: 1) The examination should not be done on blood taken within 48 hours after a rectal examination since this causes the release of prostatic ACP into the circulation.
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2) The prostatic isoenzyme is unstable hence assay should be done immediately after sample collection. If this is impossible, plasma or serum should be obtained and frozen. 3) Straining at stool in patients with a tendency to constipate can cause the release of prostatic ACP into the circulation. Enquires must thus be made about bowel habit and constipation treated if necessary. Total plasma ACP activity is also most often raised due to the presence of haemolysis. It may also be increased in some diseases affecting liver and bone to such an extent that plasma ACP activity is greatly elevated. In these cases the enzyme activity is partly tartrate-stable and may thus be distinguished from prostatic ACP. An increase in tartrate-stable ACP is also found in Gauchers disease (disorder of lipid metabolism:-lipid reticulosis).

B. THE PROSTATE SPECIFIC ANTIGEN The prostate specific antigen (PSA) is an enzyme a serine protease that is normally confined to the epithelial tissue of the prostate gland. It has a molecular weight of 33-34 kDa. The molecule consists of approximately 92% peptides and 8% carbohydrates. PSA exists in at least 5 isomers with isoelectric points (pI) ranging from 6.8 to 7.5. The major isoform having a pI of 6.9. Like other proteases, PSA in serum consists mostly of complexes with inhibitors. The predominant complex is with alpha-1 anti-chymotrypsin. Smaller amounts of PSA are also bound with -1-trypsin inhibitor, -2-macroglobulin and inter--trypsin inhibitor. A minor amount of PSA also exists in the free form but whether the free form is biologically active remains to be determined. Most of the commonly available immunoassay kits for PSA appear to detect both the free PSA and the ACT complex. The sum of both fractions contributes to the total PSA. As a serine protease, PSA can catalyze the hydrolysis of a number of proteinicious substances. Measurement of serum of PSA provides a very sensitive index of prostate cancer. Being increased in over 90% of cases when first diagnosed; in only about 50% of cases have increased serum prostatic acid phosphatase (PAP) activity at this stage. However serum PSA is increased in most patients with benign prostate hypertrophy, so the measurement lacks specificity as a test for cancer. In many western countries prostate cancer is the second most common malignancy in men. One potential approach to reducing the mortality from prostate malignancy is to screen asymptomatic men for early disease employing the PSA Kit. Using, this approach prostate cancer could be detected when it is confined to the prostate and therefore potentially curable. The enzyme of choice in the diagnosis of metastatic carcinoma of the prostate is ACP, an enzyme which hydrolyzes organic phosphomonoesters at a pH optimum of 5-6. Isoenzymes of this enzyme are found in the prostate, liver, spleen, red cells and platelets. These isoenzymes may be distinguished from one another by measuring activity in the presence of selective inhibitors. The activity of the prostatic isoenzyme is almost completely inhibited in the presence of L (+) tartrate. It is however stable in the presence of formaldehyde.

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The following must be observed when investigating prostatic disease by means of ACP: The estimation should not be performed on blood taken within 48 hours of a rectal examination. Owing to the instability of the prostatic isoenzyme the specimen must be sent to the laboratory immediately after collection. If this is impracticable, the plasma or serum should be frozen until analysis is performed. Patients who strain at stool must be treated before analysis since this elevates the activity of ACP. 6. ACUTE PANCREATITIS Assays of serum amylase, lipase and trypsin are applied to the investigation of pancreatic disease. The most important of them, however, is the measurement of alpha amylase. Amylases are a group of hydrolases that split large polysaccharides consisting of -D-glucose units. Both linear polyglucans such as amylose and branched polyglucans such as amylopectin and glycogen can be hydrolyzed. Two types of amylases have been identified: 1. The -amylase (e.g. plant and bacterial exoamylase) which acts only at the terminal reducing end of the polyglucan chain, splitting off 2 glucose units at a time. 2. amylases also called endoamylases are the human amylases. They attack the -1, 4- linkage in a random manner anywhere along the polyglucan chain. Big polysaccharides can thus be broken down into small units (dextrins, maltose and some glucose). The optimum pH for amylase activity is 6.9-7.0. It is usually assayed at 37oC although it is active at 50oC. Alpha amylase has several isoenzymes, the most abundant is derived from the pancreas where it is synthesized by the acinar cells and then secreted into the intestinal tract by way of the pancreatic duct system. A potent amylase is secreted by the salivary glands. This isoenzyme initiates hydrolyzes in the mouth and esophagus. The action of the salivary isoenzyme is terminated by acid in the stomach; then pancreatic and intestinal isoenzymes of amylase breakdown the polyglucans into maltose. Intestinal amylase breaks the maltose to glucose which is then absorbed into the circulation. In acute pancreatitis, serum levels of the enzyme rise 2-12 hours after the onset of acute pancreatitis in many patients and within 24 hours in about 85% of cases. In most patients the serum amylase level reaches a peak by 24hours and returns to normal in 48-72 hours. The administration of glucose causes a decrease in serum amylase level. Thus blood for amylase determination should not be taken during an intravenous infusion containing glucose. Also blood for serum amylase measurement should be collected at least 1 hour and preferably 2 hours after the patient has eaten. Causes of Increased Serum Amylase: > 10X upper limit of normal: - acute pancreatitis > 5X upper limit of normal: - perforated duodenal, ulcer, intestinal obstruction, other acute abdominal disorders, acute oliguric renal failure, diabetes ketoacidosis. Usually < 5X upper limit of normal:- salivary gland disorders e.g. calculi and inflammation (including mumps), chronic renal failure, macroamylasaemia, morphine administration (spasm of sphincter of Oddi)

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7. SUXAMETHONIUM SENSITIVITY There are some subjects who relapse into prolonged respiratory paralysis (Suxamethonium or Scoline Apnoea) after the use of suxamethonium, a muscle relaxant. Although these patients and their relatives often have a low pseudo-cholinesterase activity, many have values within the reference range, thus making it impossible to diagnose this condition solely on the basis of pseudo-cholinesterase activity measurements. The diagnosis can be effectively made by activity measurements in the presence of dibucaine, a spinal anaesthetic. The percentage inhibition of pseudo-cholinesterase by dibucaine is called the dibucaine number. Whereas the normal enzyme is markedly inhibited, the abnormal enzyme is only slightly inhibited by dibucaine. The population may be classified into three groups as follows:Dibucaine Number 1. Normal Zygote 75 85 2. Heterozygote 45 70 3. Homozygote 15 3 NON- PATHOLOGI CAL CAUSES OF RAISED ENZYME LEVEL 1) The level of creatine kinase is usually raised after muscular exercise and after muscular injection. 2) Physiological conditions a. Newborn the level of AST is moderately high in neonates. b. Childhood - the level of alkaline phosphatase is usually high until after puberty. c. Pregnancy in the last trimester, levels of alkaline phosphatase are raised. There are moderate rises of several enzymes such as transaminases and creatine kinase during and immediately after labour. 3) Drug induction Some drugs particularly diphenylhydantoin and the barbiturates may stimulate the production of microsomal enzymes Induction. Fro example, raised levels of gamma Glutamyl transferase (GGT) in patients taking them does not necessarily imply a pathological condition 4. Nature of sample Haemolysed samples are unsuitable for enzyme estimation. Even though the red cells may not contain the enzymes to be measured, other released enzymes may interfere with the estimation thus giving falsely elevated results. NOTE: These factors introduce a considerable difficulty in establishing the uppers limit for reference values of the various enzymes. The prediction of a condition may be improved by: a. The determination of the activities of various isoenzymes b. The estimation of more than one enzyme c. Clinical observation.

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