DIAGNOSTIC ENZYMES

INTRODUCTION Enzymes are the biological catalysts. Assay of enzymes present in blood plasma or

serum have been routinely carried out in clinical chemistry laboratories

Diagnostic enzymes refers to the enzymes that are used directly or as components of the assay system for the determination of number of substances

Changes in the concentrations of various biomolecules are indications of abnormal metabolic activities,infections,infectious and non-infectious diseases and inflammatory conditions

• Use to detect and quantify certain substances

• As labels in enzyme immuno assay (EIA) system

• There are many alternative techniques which are routinely used for the diagnosis by clinical laboratories and include Electrophoresis, chromatographic techniques,isoelectric focusing etc

LACTATE DEHYDROGENASE Important enzyme found throughout the body and

involved in glucose metabolism Tetramer of 2 different subunits(H or M) LDH1 is found predominantly in heart muscle and in

RBCs.Most stable and runs the furthest in electrophoresis strip.

LDH5 found in liver and skeletal muscle is the least stable and runs the shortest on electrophoresis

LDH3 is found in a variety of tissues such as spleen,lung,endochrine glands and lymph nodes

The analysis of isoenzyme patterns can help in the investigation of myocardial infarctionFlipped LDH isoenzyme pattern

If plasma is subjected to electrophoresis at pH 8.6,the LDH isoenzymes may then be located by means of specific stain-a mixture of lactate,NAD+ and a chromogen which will form a colored productNormal value-180-360U/L

ASPARTATE TRANSAMINASE These enzymes are found in most tissues

through out the body ,but especially in skeletal muscle, cardiac muscle, liver and kidney.

It is formally known as glutamate oxaloacetate transaminase(GOT).

Useful in the diagnosis of myocardial infarction. Elevated AST levels is indicative of damage to the myocardium.

Normal range-male 35<U/L,female31<U/L

ALANINE TRANSAMINASE Formally known as glutamate pyruvate

transaminase(GPT)

Found in high concentrations in liver cells and in much smaller concentrations elsewhere.

Hence a markedly raised plasma activity indicates a severe liver disease, usually viral hepatitis or toxic liver necrosis

Normal values-male<45U/L,female <34U/L

ALKALINE PHOSPHATASE High levels are found in liver,bone,placenta and

intestine

Used as a marker of cholestatic liver disease

Separation by electrophoresis and also assessed using inhibitors, specific substrates or by heat inactivation of other isoenzymes

• Can be visualized with a specific stain such as calcium a-naphthyl phosphate mixed with diazonium salt• Placental ALP is very heat-stable whereas liver and bone isoenzymes are much less stable at elevated temperatures• Hence the effect of heat on the total enzyme activity can sometimes help to indicate which are the predominant isoenzymes present

Sex Age Normal range(U/L)

Male/female 4-15 54-369

Male

females

20-50≥60

20-50≥60

53-12856-119

42-9853-141

CREATINE KINASE Also known as creatine phosphatase(CPK)

Mainly found in heart and skeletal muscle and in brain

It is a dimer made up of 2 types of polypeptide chains(B orM) in any combination. Thus 3 isoenzymes are found:BB,the main form of brain,MB-in heart muscle and diaphragm and MM-both heart and muscle

• Electrophoresis , immunological techniques and trazine dye affinity chromatography are useful in the analysis of isoenzymes

• Increased plasma activities of CK(MM+MB) results in severe damage to heart cells.

• Normal range=male-46-171U/L,female-34-145U/L

ACID PHOSPHATASE Maximum activity-pH-5-6

Found in large amounts in prostate glands and its assay in plasma has been used in the diagnosis of prostatic carcinoma

Also found in liver, red cells, platelets and bone. They are analyzed by immunoassay techniques or by the actions of inhibitors. The prostate and red cell forms of enzymes are inactivated by ethanol, red cell form by formaldehyde, and the prostate form by L-tartarate

Normal range-0.1-0.4U/L

CHOLINESTERASE The enzyme catalyze the hydrolysis of choline esters

Presence is assumed when the muscle relaxant scoline is administered. Patients with low plasma activities of cholinesterase experience severe breathing difficulties for several hours after treatment with scoline, so a preliminary assay may be carried out to see if it safe to administer the drug.

Normal range=male-40-78U/L,female-33-76U/L

Α-GLUTAMYLTRANSFERASE(GGT,Α-GT) Found in biliary ducts of the liver, in the kidney and

pancreas with the largest amounts being in kidney Also found in hepatocytes where its enzyme activity

can be induced by a number of drugs and in particular alcohol, thus making GGT,a useful marker of alcohol induced liver disease and in particular liver cirrhosis.

Normal range=male-<55U/L,female-<38U/L

AMYLASE Found in high concentrations in pancreas and

salivary glands where it is secreted to digest complex carbohydrates

Useful in those patients with acute abdominal pain, to differentiate between patients with acute pancreatitis and those with appendicitis

Patients with acute pancreatitis will have high levels of amylase in their blood

Normal range=28-100U/L

INBORN ERRORS OF METABOLISM Another application is in the investigation of an

inborn error of metabolism.

In such conditions, the enzyme is not fully active, resulting in a pathological condition called an inherited disorder.

The activity is measured in blood and sometimes in preparation of cells such as fibroblast

Disorder Enzyme affected

Phenylketonuria Phenylalanine hydroxylase

Alkaptonuria Homogenitisic acid oxidase

galactosaemia galactokinase

Von gierke’s disease Glucose-6-phosphatase

Tay-sachs disease Β-N-Acetyl glucosaminidase A

Hurler’s syndrome A-L- Iduronidase

ENZYMES AS REAGENTS IN CLINICAL CHEMISTRY D-glucose in blood and other physiological fluids is

commonly analyzed by means of procedures involving glucose oxidase.

The reaction catalyzed by glucose oxidase is utilized for the screening of urine specimens.

Used in the diagnosis of diabetes Mellitus Blood cholesterol may be determined by a reaction

catalyzed by cholesterol oxidase, the product 4-cholesten-3-one having an absorption maxima at 240nm.

IN FORENSIC MEDICINE For detecting body fluids and as a marker of genetic

individuality Test for seminal acid phosphatase activity Used in DNA finger printing Forensic markers-adenosine deaminase,adenylate

kinase,carbonic anhydrase,esterase-D,glucose-6-phosphate dehydrogenase,peptidase-A,glyoxalase,phosphoglucomutase