Enzyme Diagnostics

Enzymes –Complex protein catalysts, found in small amounts, cause specific chemical change in all parts of the body. Present in all parts of the body including: blood, intestinal fluids, mouth (saliva),stomach (gastric juice).

-Normally only small quantities of intracellular enzymes leak from cells into blood or other body fluids.

Serum- is the component that is neither a blood cell (serum does not contain white or red blood cells) nor a clotting factor; it is the blood plasma with the fibrinogens removed. Serum includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens and hormones.

Enzyme and Serum

Sources of non-functional enzymes

•Cell damage

•Obstruction of normal pathways: eg obstruction of bile duct increases alkaline phosphatase

•Increase of the enzyme synthesis: eg Bilirubin increases the rate of synthesis of alkaline phosphatase in obstructive liver disease.

•Increased permeability: eg hypoxia.

Medical importance of Non-functional enzymes

Measurement of these enzymes is important for:

•Diagnosis of diseases- as disease of different organs cause elevation of different plasma enzymes.

•Prognosis of the disease- Follow up treatment pre and post measurement of enzymes

Serum Enzymes used for diagnostics

MYOCARDIAL INFARCTION

A heart attack -blood flow to a part of your heart is blocked ,part of theheart muscle is damaged or dies. The medical term for this is Myocardialinfarction.

SYMPTOMS

• Chest pain- Pain restricted or may move from chest to your arms, shoulder, neck, teeth, jaw, belly area , back.Pain usually lasts longer than 20 minutes.

Other symptoms-

• Anxiety

• Cough

• Fainting

• Light-headedness, dizziness

• Nausea or vomiting

• Palpitations (feeling like your heart is beating too fast or irregularly)

• Shortness of breath

• Sweating (which may be very heavy)

CAUSES:

• Blood clot that blocks one ofthe coronary arteries. Thecoronary arteries bring bloodand oxygen to the heart. If theblood flow is blocked, theheart is starved of oxygen andheart cells die.

• Plaque-A hard substance canbuild up in the walls of yourcoronary arteries. This plaqueis made up of cholesterol andother cells.

• The most common cause of blocked arteries is atherosclerosis. Exact cause of atherosclerosis is unknown, but most researchers believe it begins with an injury to the innermost layer of the artery, known as the endothelium.

The following factors are thought to contribute to the damage:• High blood pressure

• Elevated LDL ("bad") cholesterol

• An accumulation of homocysteine (amino acid produced by the human body,

thought to be a risk factor for heart disease, stroke, osteoporosis, diabetes, and

dementia)

• Smoking

• Diabetes

• Inflammation

• This is a normal coronaryartery with a nice, big,unobstructed lumen forsupplying plenty of bloodto the myocardium.

• At the left the lumen is about 50% occluded. At the right, there has been thrombosis with organization and recanalization to leave three small remaining lumens.

• This coronary artery demonstrates yellowish atherosclerotic plaques grossly.

• Coronary artery opened longitudinally demonstrates severe atherosclerosis.

• 75% narrowing• Complete occlusion.

TESTS1.Troponin test

• Composed of three regulatory proteins (troponin C, I and T)

• Attached to tropomyosin ,found in the grooves between actin filaments in muscle tissue.

• Found in skeletal and cardiac muscle but not smooth muscle.

• Troponin I and T- Excellent markers for myocardial infarction

• Serum levels of troponin I rise within 4-8 hrs after the onset of chest pains ,levels peak within 12-16 hrs ,return to baseline within 5-9 days.

• Measurement of serum LDH fractions was once considered the ideal marker for onset and severity of a heart attack, the high specificity of troponin I to heart muscle necrosis make this protein the preferred marker to measure in patients suspected of suffering a myocardial infarct.

• Normal Values• Cardiac troponin levels are normally so low they cannot be detected with

most blood tests.• The test results are usually considered normal if the results are:• Troponin I : less than 10 µg/L• Troponin T : 0–0.1 µg/L• Normal troponin levels 12 hours after chest pain has started mean a heart

attack is unlikely.

• What abnormal results mean• Even a slight increase, usually means there has been some damage to the

heart. Significantly high levels of troponin are a sign that a heart attack has occurred.

• Most patients who have had a heart attack have increased troponin levels within 6 hours. After 12 hours almost everyone who has had a heart attack will have raised levels.

• Troponin levels may remain high for 1 to 2 weeks after a heart attack.

Increased troponin levels may also be due to:• Abnormally fast heart beat

• High blood pressure in lung arteries (pulmonary hypertension)

• Blockage of a lung artery by a blood clot, fat, or tumor cells (pulmonary embolus)

• Congestive heart failure

• Inflammation of the heart muscle usually due to a virus (myocarditis)

• Strenuous exercise (for example, due to marathons)

• Trauma that injures the heart such as a car accident

• Weakening of the heart muscle (cardiomyopathy)

• Cardiac angioplasty/stenting

• Purposeful shocking of the heart by medical personnel

• Open heart surgery

2.Creatine Kinase (CK)/Creatine Phosphokinase (CPK)

• Creatine Creatine Phosphate

ATP ADP

(Energy source for muscle contraction)

• Dimer (consists of 2 protomers)-B (after brain) and M (after Muscle).

• 3 Tissue specific isoenzymes:

• CPK1 (CPK-BB) is the characteristic isoenzyme in brain and is in

significant amounts in smooth muscle and is 0% of the normal serum total.

• **It increases in brain tumors.

• CPK2 (CPK-MB) accounts for about 35% of the CPK activity in cardiac

muscle , but less than 5% in skeletal muscle and is 0% of the normal serum

total.

• **It increases in heart diseases.

• CPK3 (CPK-MM) is the predominant isoenzyme in muscle

and is 100% of the normal serum total.

• **It increases in skeletal muscle diseases

• Found in heart, skeletal muscle, brain.

• Level of CPK rises within 6hrs and peak by ~18hrs.

• If not persistent, normal within 2-3 days.

• CPK-MB levels rise 3-6 hrs after a myocardial infarction

and peak 12-24 hrs later, returns to normal 12-48 hrs after

the infarct.

• Normal Values:

male: 5-35 ug/ml (mcg/ml);female: 5-25 ug/mlnewborn: 10-300 IU/L

• Precautions:

• Draw the sample before giving or one hour after giving I.M. injections. I.M. injections will increase the total CK level.

• Obtain the blood samples on schedule. Always note the time the sample was drawn and the hours elapsed since onset of chest pain.

• Be sure to handle the sample gently to prevent hemolysis. Always have the sample transported to the lab promptly because CK activity diminishes significantly after 2 hours at room temperature.

3.Lactate dehydrogenase(LDH)

Lactic acid Pyruvic acid

NAD NADH+H

• LDH is a tetramer(consists of 4 protomers)-H(after heart) and M(after

muscle)

• LDH has 5 isoenzymes:-• LD1 and LD2 -heart, red blood cells and kidneys. • LD3 –lungs• LD4 and LD5 -liver, skin, and skeletal muscles.

• LDH elevates in 24-48 hours and peaks in 48-72 hours after the episode.• Narcotic drugs and IM injections can elevate serum LDH levels. Hemolysis

of the blood can cause an elevated LDH because LDH is plentiful in the erythrocytes.

• Because many common diseases increase total LDH (LD) levels, isoenzyme electrophoresis is usually necessary for diagnosis

4.SGOT

• Serum Glutamic Oxaloacetic Transaminase called: AST, (Aspartate Aminotransferase) (is released with tissue necrosis).

• Aspartate (Asp) + α-ketoglutarate ↔ oxaloacetate + glutamate (Glu)

• Is an important enzyme in amino acid metabolism.

• AST is found in the liver, heart, skeletal muscle, kidneys, brain and red blood cells.

• Normal Values: 5-40 U/ml (Frankel) 4-36 IU/L; or 16-60 (Karmen) U/ml U/L at 30 degrees C; or 8-33 (SI units) at 37 degrees C.

• Clinical Implications:

• Elevation 8-12 hours after infarction. Peak levels are reached 24-48 hoursafter the MI.

• This enzyme is not particularly indicative of an MI. Other conditions can also cause a rise in the levels. High levels of SGOT may be obtained with trauma to the skeletal muscles, in liver disease, pancreatitis and others.

• *Decreased levels of enzyme are found in pregnancy, diabetes, beriberi. Elevations can be caused by hepatitis, trauma, musculoskeletal disease, IM injection, pancreatitis, liver cancer, and strenuous exercise.

5. SGPT

• Serum Glutamic Pyruvic Transaminase- enzyme is called Alanine

Transaminase (ALT)

• α-ketoglutarate + L-alanine ⇌ L-glutamate + pyruvate

• ALT is particularly diagnostic of liver involvement as this enzyme is found

predominantly in hepatocytes.

• Elevated levels of ALT (SGPT) = viral hepatitis, diabetes, congestive heart

failure, liver damage, bile duct problems or myopathy.

• ALT is commonly used as a way of screening for liver problems.

• Elevated ALT may also be caused by dietary choline deficiency.

• Fluctuation of ALT levels is normal over the course of the day, and ALT

levels can also increase in response to strenuous physical exercise.

• Normally in liver disease or damage that is not of viral origin the ratio of ALT/AST is less than1.

• With viral hepatitis the ALT/AST ratio will be greater than1.

• level of AST elevation in the serum =number of cells involved

• Following injury, levels of AST rise within 8 hours and peak 24–36 hours later. Within 3–7 days the level of AST should return to pre-injury levels unless further injury occurs.

* Although measurement of AST is not, in and of itself, diagnostic for myocardial infarction, taken together with LDH and CK measurements the level of AST is useful for timing of the infarct

• Preventive Care: You can reduce your risk of heart attack by:

• Quit smoking.• Aerobic exercise (such as walking, biking, or swimming) for at least 30

minutes 5 days per week.• Reducing stress and learning stress-reduction techniques such as deep

breathing and meditation. • Eating a diet low in saturated fat and rich in fruits, vegetables, and whole

grains.• Losing weight or maintaining a proper weight.• Follow your doctor’s instructions to keep these risk factors under control.

• Treatment Approach:

• The goal when treating a heart attack is to restore blood flow to the affected area of the heart immediately, to preserve as much heart muscle and heart function as possible. If your doctor has prescribed nitroglycerin, take it while you are waiting for emergency medical personnel to arrive.

• Medications

• When you arrive at the hospital, you will likely be given one or more

medications to help your body cope with, or ward off, damage from the

heart attack, including:

• Aspirin -- helps stop blood from clotting. You may be given aspirin in the

ambulance or as soon as you get to the hospital. Aspirin should be

continued indefinitely at a dose of 81 mg per day.

• Nitroglycerin -- helps dilate (widen) blood vessels. You may be given

nitroglycerin in the ambulance or as soon as you get to the hospital.

• Pain reliever -- helps relieve pain and is often given intravenously (IV).

• Thrombolytic -- work to break up clots. They are most effective when

taken within 2 hours of the heart attack, and are not given after 12 hours

have elapsed. These drugs may be given with other anticoagulants (blood

thinners).

• Anticoagulants (blood thinners) -- make your blood less likely to form

clots. Heparin is often given by injection while you are in the hospital.

• The prostate gland, which isassociated with theproduction of sperm in men,secretes an enzyme calledprostatic acid phosphatase(PAP). In all stages ofprostate cancer, except thefinal stages, PAP levels areraised compared to those ofhealthy individuals.

• Hydrolase enzyme -catalyze the hydrolysis of a chemical bond. Specifically, it targets and breaks the molecular bonds of phosphate groups.

• ACP can be found in certain organs and tissues, including blood cells, bone marrow, the spleen, pancreas, liver and kidneys.

• Found in the greatest concentration in the prostate and up to 1,000 times greater in seminal fluid than any other bodily fluid.

• The latter fact is useful in forensics since the detection of prostatic acid phosphatase (PAP) in vaginal tissue measuring levels greater than 3 U/ML (units per milliliter) is used as supporting evidence that a rape has occurred.

• It is stored in lysosomes and functions when these fuse with endosomes, which are acidified while they function; therefore, it has an acidic pH.

• Normal Value:0 to 1.1 Bodanzky units/ml;1 to 4 King-Armstrong units/ml;0.13 to 0.63 BLB units/ml.

ACID PHOSPHATASE

• Clinical Implications:

• Appears primarily in the prostate gland and semen. It is also found in other organs, but in very small amounts. Prostatic and erythrocytic enzymes are the two major isoenzymes.

• The prostatic isoenzyme is more specific for prostatic cancer. widespread the tumor=produce high serum acid phosphatase levels.

• Marked increased acid phosphatase levels: A tumor that has spread beyond the prostatic capsule

• Moderately increased acid phosphatase levels: Prostatic infarction, Paget's disease, Gaucher's disease, multiple myeloma

• Declining high acid phosphatase levels: Successful treatment of prostatic cancer

• Hemolysis due to rough handling of sample or improper storage may interfere with test results.

• Acid phosphatase levels drop by 50% within one hour if the sample stays at room temperature without the addition of a preservative or if it is not packed in ice.

• Abnormal levels may indicate the presence of systemic infection, anemia, hepatitis.

• Elevated levels of PAP in particular is associated with inflammation of the prostate, as well as prostate cancer that has metastasized to the bone.

• Phosphatase testing is also used to assess enzymatic damage caused by kidney disease, liver disease, or a heart attack.

• The blood sample is exposed to one of several agents to elicit an enzymatic reaction, namely 4-aminoantipyrine, or a solution of disodium phenyl phosphate and citrate.

• To measure levels of PAP, tartrate is used. By observing the reaction with these different agents, the clinician is able to determine which tissue is releasing phosphatase enzymes into the bloodstream and what type they are. Usually, the test results are available within one to two days.

• The diagnostic precision of plasma enzyme analysis may be improved by:

• 1. Estimation of more than one- For instance, although both alanine andaspartate transaminases are abundant in the liver, the concentration ofaspartate transaminase is much greater than that of alanine transaminase inheart muscle

• 2. Isoenzyme determination- Some enzymes exist in more than one form:these isoenzymes may be separated by their different physical or chemicalproperties. For example, creatine kinase may be derived from skeletal orcardiac muscle, but one of its isoenzymes is found predominantly in themyocardium

• 3. Serial enzyme estimations- The rate of change of plasma enzymeactivity is related to a balance between the rate of entry and the rate ofremoval from the circulation. A persistently raised plasma enzyme activity issuggestive of a chronic disorder or occasionally of impaired clearance.

• Factors Affecting Results of Plasma Enzyme Assays

1. Analytical factors affecting results:

• The total concentration of all plasma enzyme proteins is less than 1 g/L. Results of enzyme assays are not usually expressed as concentrations, but as activities.

• The results of such measurements depend on many factors. These include the concentrations of the substrate and product, the pH and temperature at which the reaction is carried out, the type of buffer, and the presence of activators or inhibitors.

• Because the definition of 'international units' does not take these factors into account, results from different laboratories, apparently expressed in the same units, may not be directly comparable. Therefore, plasma enzyme activities must be interpreted in relation to the reference ranges from the issuing laboratory.

2. Physiological factors affecting enzyme activities include for example:

a. Age: Plasma AST activity is moderately higher during the neonatal period than in adults, plasma alkaline phosphatase activity is higher in children than in adults and peaks during the pubertal bone growth spurt before falling to adult levels.

b. Sex: plasma γ-glutamyl transferase activity is higher in men than-in women.

c. physiological conditions: Plasma alkaline phosphatase activity rises during the last trimester of pregnancy because of the presence of the placental isoenzyme: several enzymes, such as the transaminases and creatine kinase rise moderately in plasma during and immediately after labour or strenuous exercise.

ThanK You!