PLASMA ENZYME DIAGNOSIS

• Enzymes are biological catalyst that speed up the rate of reaction

1. Essential reactions catalyse by enzymes

2. Hydrolysis

3. Esterification

4. Synthesis

5. Molecular inter-conversions

• These rxns are essential for the supply of energy and or chemical changes necessary for body vital activities such as:

• Nerve conduction

• Muscle contraction

• Digestion

• Growth

• Respiration

• Reproduction

• Body temperature maintenance, etc

FACTORS AFFECTING ENZYME REACTION

• Concentration of reactants

• pH

• Temperature

• Ionic strength

• Presence of specific ions(activators)

• Presence of inhibitors

• Assay of the enzyme activity must be carried out under optimum condition for the substrate concentration, pH, buffer selection and conc, coenzyme, activators, temperature, and time of incubation.

• Differences in the reference values of enzyme activity are due to changes in assay conditions from one clinical laboratory to another, since reference values are assay method dependent.

• There are three major classes of enzymes in the bloodstream or plasma.

• These are:

• The functional enzymes

1. Pseudocholinesterase

2. Lipoprotein lipase

3. Caeruloplasmin

4. Coagulation factors

5. Complements

6. Renin and other plasma enzymes

• The digestive enzymes

• The intracellular enzymes are clinically useful when serum or plasma concs or level are increased above or decreased below the normal range

ISOENZYMES

• These are different forms of an enzyme that differ in few amino acids. The multiple natural forms of an enzyme, which catalyse the same reaction in a single species, are isoenzymes. Isoenzymes are different proteins with similar enzymatic or catalytic activity. Even though they catalyses the same rxns they may have different Km values and may be inhibited by different substances or compounds(e.g. alkaline phosphatase, acid phosphatase,lactate dehydrogenase, etc)

• Isoenzymes can be demonstrated by:

•

• Gel electrophoresis

• Isoelectric focusing

• Column chromatography

• Physical ppt

• Chemical structures

• Kinetics

• Immunological reaction with antisera

PLASMA ENZYME MEASUREMENTS OF DIAGNOSTIC IMPORTANCE

• Lactate dehydrogenase – the enzyme reversibly catalyses the oxidation of lactate to pyruvate by transferring hydrogen from lactate to the cofactor NAD+.

• It is tetramer. The subunits are of two forms which are designated H and M.

• Have 5 isoenzymes

• Lactate dehydrogenase is found in the cytosol of all human cells and thus may have little diagnostic specificity.

• Total LDH activity in plasma may be increased in a wide number of disorders which includes

• Artefactual – due to in-vitro haemolysis or delayed separation of plasma from whole blood.

• Marked increase – in circulatory failure with shock and hypoxia, myocardial infarction, some hematilogical disorder

• Moderate to slight increase – viral hepatitis, malignancy of any tissue, skeletal muscle disease, pulmonary embolism, infectious mononucleosis, certain drugs.

• Creatine kinase (CK) – catalyses the reversible transfer of phosphate from adenosine triphosphate to creatine. The enzyme reaction makes it possible for the storage of high density energy phosphate in a more stable form than in ATP.

• Has three principal isoenzymes

• CK-1, in the brain

• CK-2, in the cardiac muscle

• CK-3, in the skeletal and cardiac muscle

• The most important is that the CK-2 serum levels increases within 4 to 6 hours after myocardial infarction and peak is about 12 to 20 hours post-infarction. It is within normal level 1 to3 days post-infarction. CK-2 is specific for myocardial damage.

• Causes of raised plasma CK activities

• Artefactual- due to in-vitro haemolysis, using most methods

• Physiological – neonatal period, during and for a few days after parturition

• Higher in africans compared to caucasians

• Marked increase – dermatomyositis and polymyositis, shock and circulatory failure,myocardial infarction, muscular dystrophies, rhabdomyolysis, necrotizing fasciitis

• Moderate to slight increase – muscle injury, infection,after surgery, physical exertion, after intramuscular injection, hypothyroidism, alcoholism, some cases of cerebovascular accident and head injury, malignant hyperpyrexia

• Aminotransferases(transaminases) – catalyses the transfer of an amino group (NH2)from an amino acid directly to another molecule without the liberation of free ammonia.

• Aspartate aminotransferase(AST) – present in most tissues, especially in skeletal and heart muscle, liver and kidney.

• Two major isoenzymes are identified: the cytopasmic isoenzyme and the mitochondrial isoenzyme.

• Both are released into the plasma during injury or damage ti the cells and they are shown to be of diagnostic value

• Increased serum level are found in

• Artefactual – due to in-vitro release from erythrocytes if there is hemolysis or if separation of plasma from cells is delayed.

• Physiological – neonatal period

• Marked increase – circulatory failure with shock and hypoxia, myocardial infarction, acute viral or toxic hepatitis

• Moderate to slight increase – cirrhosis, infectious mononucleosis,cholestatic jaundice,malignant infilteration of the liver, skeletal muz disease, after trauma or surgery, severe haemolytic episodes.

• Alanine aminotransferase(ALT) – predominanantly found in the liver, but significant amount is present in kidney.

• ALT is more specific for liver disorders than AST

• Plasma level may be used to distinguishing btw hepatocellular jaundice from obstructive jaundice since ALT shows obvious increase in hepatocellular jaundice and only weak increases in obtrusive jaundice

• It is mildly increased in other disease conditions which include:

1. Acute renal disease

2. Muscle disease

3. Disseminated carcinoma

• Phosphatases – this is a common name for a group of enzymes, which hydrolyse the phosphate esters with little specificity for the preferred substrate

• They can be divided into

• Acid phosphate – mostly active at approx pH 5. found widely in human tissues:

• Prostate, bone, kidney, erythrocytes, platelets, liver, spleen

• The prostate is the richest source of this enzyme.

• Acid phosphatase measurements are very important for the diagnosis and monitoring therapy of carcinoms of the prostate, incuding metastatic and invasive forms of the carcinoma

• Present in seminal fluid and this has been useful forensically in the diagnosis of rape, as vaginal acid phosphatase is very low

• Increased plasma acid phosphatase occurs in:

I. Prostatic hyperplasia

II. Prostatic inflammation

III. Prostatic trauma

IV. Prostatic necrosis

• Tartrate-stable(non-prostatic) isoenzyme of acid phosphatase often increases in:

• Bone disease, esp paget’s disease, hyperparathyroidism, metastatic breast carcinoma

• Plasma total acid phosphatase may also be increased in the ffg

• Liver disease

• Gaucher’s didease

• Thrombocytopaenia(when it is due to platelet lysis)

• Alkaline phosphatase

• Physiological changes

• In normal pregnancy release from placenta

• In infancy and childhood, which is due to osteoblastic activity of the normal growing bones

• Faty meal may stimulate the release of intestinal alkaline phosphatase.

• Fasting blood sample are preferred for the measurement

• Clinical application of plasma alkaline phosphatase is very important in:

• Liver disease

• Bone disease

• Tumour marker

• 5’-nucleotidase – a specific alkaline phosphatase. It is a phosphatase enzyme that splits off the phosphate from 5’-adenosine monophosphate. Found in liver associated with canalicular and sinusoidal plasma memebranes.

• It is released into the plasma only as a result off the disease of the liver and biliary trees. Used clinically to distinguish btw bone or liver increases in plasma or serum alkaline phosphatase

• It is of significant value than alkaline phosphatase in the diagnosis of obstructive jaundice in paediatrics. Since its increased level do not originate from the bone. It is also not elevated in pregnancy

• Gamma-glutamyltransferase(GGT) – involve in the transfer of gamma-glutamyl from gamma pepetides to amino acids, H2O, and other small pepetides. Glutathione serves as the gamma-glutamyl donor in most biological media.

• The enzyme occurs mainly in the liver, kidney and pancreas but occurs in particularly high conc. In the epithelial cells lining the biliary ductules. Also found within the hepatic parenchyma, in the smooth endoplasmic reticulumand as such subject to liver microsomal induction. Plasma elevated increases are however from the liver

• Increase may be seen in cholestatic liver disorders(ie obstuctive jaundice), it may also be due to drug induction in the absence of clinical evidence of liver disorder in patient treated with

• Phenytoin

• Warfarin

• Henobarbitone and other barbiturates

• Some other drugs due to liver enzyme induction

• Due to hepatotoxins

• Chronic alcoholics

• It is the most sensitive marker of acute alcoholic hepatitis

• Alpha-amylase – produced by the salivary and pancreatic glands into their resp. juices. They are released into the GIT and essential for the digestion of starches.

• In normal plasma both S-type alpha-amylase and p-type alpha-amylase are present. Partially filtered in the glomerulus because of their small size

• Increase plasma level occur in

• Acute pancreatitis

• Acute abdominalconditions

• Salivary gland disease, acute IT of this gland(mumps)

• Ectopic pregnancy

• Malignant tumours

• Renal disease

• drugs

• Urine and serum amylase activities are used in the diagnosis of acute pancreatitis.

• Lipase – is a single polypeptide chain glycoprotein and a small molecular weight of about 48,0000 Daltons. The presence of bile salts and cofactor is essential for full lipase activity. It hydrolyses emulsified triglycerides. Secreted by the exocrine gland of the pancreas and it is produced in the pancreatic juice along with other digestive enzymes.

• Lipase is measured along with amylase because it adds sensitivity and specificity to the diagnosis of acute pancreatitis. Has longer half life than amylase.

• Increase plasma activity is seen in

• Duodenal ulcer

• Perforated peptic ulcers

• Intestinal obstruction

• Acute cholecystitis

• Cholinesterase – hydrolses choline esters(acetycholine). There are two types acetycholinesterase and pseudocholinesterase.

• The plasma cholinesterase is the pseudocholinesterase produce chiefly in the liver

• Analyses of serum pseudocholinesterase activity are used for

• Liver function test

• As a marker of possible insecticide poisoning, and

• As an indicator for detecting patients with abnormal variants of the enzyme

• Also used in monitoring patients exposed to organo-phosphorus insecticides and phosphate-rich fertilizers

• Angiotensin-converting enzyme

• A dipeptidyl carboxypepetidase. Release angiotensin 1 and bradykinin. Major site is in the pulmonary endothelium.

• May be used as a marker of disease activity in sarcoidosis. It can be elevated in other lung condition and therefore is not specific for sarcoidosis.

• Tryptase

• Is a serine protease found in mast cells, in cases of mast cell degranulation, as occurs in systemic anaphylaxis, tryptase levels rise within 1 hour and remain elevated for 4-6 hours.

PLASMA ENZYME PATTERNS IN DISEASE

• Myocardial infarction – diagnosis is usually made on the clinical presentation and electrocardiographic findings and confirmed by characteristic changes in plasma enzyme activities or troponins.

• Liver disease -