ENZYMES IN DIAGNOSIS AND PROGNOSIS

GEETA JAISWALM.L.N. MEDICAL COLLEGE

ALLAHABAD

Clinical Enzymology1. Clinical enzymology is the application

of the science of enzymes in the treatment and diagnosis of diseases.

2. Measurements of the activity of digestive enzymes in the body fluids as an aid to diagnosis dates back to the early 1900s (Amylase in urine was first studied by Wohlgemuth in 1908)

3.Measurements of enzymes activity in serum began in the 1920s and 1930s.

4. The scientists who began these studies were Kay, King, Bodansky and Roberts in their work on Alkaline phosphatase.

5. In 1955 La Due, Wroblewski and Karmen reported the rise of Asparate after acute myocardial infarction from this period a great stimulus was received in the measurement of cellular enzyme released into the plasma as a consequence of tissue damage.

This observation marked the beginning of the modern phase of diagnostic enzymology.

Clinical chemists are principally concerned with changes in the activity in serum or plasma enzymes that are predominantly intracellular.

CLINICAL SIGNIFICANCE OF SERUM ENZYME

To investigate and interpret changes in serum enzymes for diagnosis.

This happens to be presently the most advancing field in clinical Biochemistry.

Enzymes in circulation are divided into two groups.

(A) PLASMA SPECIFIC OR FUNCTIONAL ENZYMES

(B) PLASMA NONSPECIFIC OR NON-FUNCTIONAL ENZYMES

(A) PLASMA SPECIFIC OR FUNCTIONAL ENZYMES

These are normally present in plasma and have specific functions to perform.

They are synthesized in the liver and enter circulation.

Their levels fall on impairment of liver or genetic disorder.

e.g. Lipoprotein lipase Plasmin Choline esterase Ceruloplasmin

(B) PLASMA NON SPECIFIC OR NON-FUNCTIONAL ENZYMES

These enzymes are either totally absent or in very low concentration in the liver, eg:

Digestive Enzymes Secretary Enzymes Enzymes associated with metabolism

Constitutive enzyme (LDH,Transaminases etc)

Enzymes in Diagnosis and Prognosis

Estimation of the activity of non-plasma specific enzymes is very important for diagnosis and prognosis of the disease

Normal serum levels indicate a balance between its synthesis and release in routine cell turnover.

Serum enzymes are used as markers to detect cellular damage, which helps in diagnosis.

Raised levels may be due to

1) Cellular damage2) Increased rate of cell turnover.3) Proliferation of cells.4) Increased synthesis of

enzymes.5) Treating patients with protein

anabolic drugs.

DIAGNOSTIC SIGNIFICANCEProvides information on the nature and

extent of the disease

Helps in diagnosis : e.g. assay of CPK helps in confirming MI even when ECG changes are doubtful.

e.g. SERUM

MI GOT LDH

Pulmonary GOT Normal LDH Embolism

Helps in ascertaining prognosis : The progress of enzyme changing process helps to ascertain the response of drugs to disease.

Helps in Early Diagnosis or Detection : When tissue changes in a disease are not pronounced enough.

Units of Enzyme Activity

Enzyme units are never expressed in terms of their concentration (as mg or µg ) but are expressed as activity.

To maintain uniformity world over enzyme activity as units is expressed according to the I.U.B system as:-

International Unit (IU) I.U or System International SI isThe activity of enzyme which

transforms one micromole of substrate per minute under optimal conditions and at a defined temperature is IU ml

IU / L = when one milli micro mole of substrate is transformed

Katal Unit

Katal Unit is a new unit. Abbreviation is KATIt is the conversion of 1 mole

substrate per second 1 IU = 60 µ katal

CLINICAL SIGNIFICANCE OF HEART FUNCTION ENZYMES

Normal serum level

Creatinine Phosphokinase (CK or CPK)

4 –60 IU/L

Aspartate Transaminase (AST, SGOT)

4 –17 IU/L

Lactate Dehydrogenase (LDH) 60 –250 IU/L

OTHER ENZYMES NOT SO COMMONLY DONE FOR HEART

FUNCTIONS

glutamyl Transpeptidase

10 –47 IU/L

Histaminase 0.12 –0.76 PU/ml

CREATININE PHOSPHOKINASE (4 – 60 IU/L)

Creatine – P + ADP Creatine + ATP CPK

Found in high concentration in skeletal muscles, myocardia and brain.

Sensitive measure for MI and Muscle diseases.

Normal in liver disease

Behavior in MI

In MI serum values rise after 6 hrs.

Peak is reached between 24 – 30 hrs.

Magnitude of elevation, greater than GOT and LDH.

SERUM GLUTAMATE OXALDACETATE TRANSAMINASE

(SGOT OR AST. (4 – 17 IU/L)OXALOACETATE + GLUTAMATE ASPARTATE + KETOGLUTARATE

• Present in high concentrations in the myocardium

• Behaviour in MI

• Rises sharply in the first 12 hrs.

• Peak at 24 hrs.

• Normal within 3 – 5 days.

ASSESSMENT OF MI THROUGH SGOT LEVELS

350 IU/L Fatal (Massive infarction)150 IU/L Associated with high mortality50 IU/L Low Mortality• Abnormal level highest on the 2nd Day.• Elevated also in Muscle disease and

Hepatic Diseases.

LACTATE DEHYDROGENASE (60 – 250 IU/L)

Catalyzes the reversible conversion of Pyruvic Acid Lactic Acid.

In acute MI, serum activity rises within 12-24 hrs.

Peak at 48 hrs.Normal from 8 – 14th Day.LDH levels persist even after CPK

and SGOT normalize.

LACTATE DEHYDROGENASE (60 – 250 IU/L)

Acute MI, LDH levels may rise as high as 1500 IU/L.

Enzyme in non-specific for myocardial tissues as LDH is widely distributed in body cells.

Co-existing disease process in other organs may cause elevation e.g. : Pulmonary infarction and renal necrosis

ENZYME RISE BEGINS PEAK NORMAL

CPK 6 hrs. 24 – 30 hrs. 72 hrs (3 days)

SGOT Rises sharply 1st 12 hrs. 24 hrs. 3 – 5 days

LDH Rise 12 – 24 hrs 24 – 48 hrs. 8 – 14 days

GLUTAMYL TRANSPEPTIDASE(10 – 4 IU/L MEN : 7 – 30 IU/L WOMEN)

Transfer glutamyl group from one peptide to the other.

Highest Tissue Activity in kidneysRelatively High Activity in liver, lungs, pancreas.

Heart normally has very little YGT.Raised serum levels in Acute MI found

later between day 7 – 11.

Useful in detecting MI in later stagesElevated levels also seen in

Heaptobilliary disorders. Alcoholics, alcoholic cirrhosis. Pancreatic diseases. In Epileptic patients.

Not elevated in any form of bone disorders.

Used to distinguish raised Alkaline phosphatase – associated with either bone or hepatic dysfunctions.

HISTAMINASE (0.12 TO 0.76 P.U /ml)

Found raised in heart muscles.Rises within 6 hrs of MI.Helps in early diagnosis of MI when ECG changes do not reveal anything. 0.8 p.u/ml is raised, 3.4 to 4 fatal.

PSEUDOCHOLINESTERASE (2.17 TO 5.17 IU/ml)

• They hydrolyze cholinesters choline.• Two types of cholinesterases are present.

(a) True cholinerterase Muscular tissue, nerve tissue,RBC.(b) Pseudo cholinesterase Heart, Intestine, Plasma.

• Provide a sensitive index for determining cellular necrosis in the myocardium.

• Raised levels as early as 3 hrs and within 12 hrs.• Levels decrease in Hepatitis and

Organophosphorus Poisoning.

SERUM ENZYMES IN LIVER DISEASESLiver functions can be ascertained by a

large numbers of enzymes.

They are divided into 2 groups

I) Most commonly and routinely done in the laboratory.

II) Not routinely done in the laboratory.

Most commonly and routinely done in the laboratory.

a) Serum Transaminasesb) Serum Alkaline

Phosphatase

II) Other enzymes not done routinelya) Serum 5' nucleotidasesb) Serum LDHc) Serum Isocitrate dehydrogenased) Serum Cholinesterasee) Ser Ornithine Transcarbamylasef) Serum Leucine Amino peptidaseg) Ser Hydroxybutyrate Dehydrogenaseh) Glutamyl Transpeptidase

SERUM ENZYMES IN LIVER DISEASESSERUM TRANSAMINASES

SGOT – 4 - 17 IU/L

SGPT – 3 - 15 IU/LSERUM ALKALINE PHOSPHATASE

3 to 13 K.A/100ml

or 23 to 93 IU/LSERUM 5’ NUCLEOTIDASES 2 – 17 IU/LSERUM LDH 60 – 250 IU/L

SERUM TRANSAMINASE

Both the enzymes are present in most tissues but relative amounts vary.

Heart Muscles are rich in SGOT.

SGOT (AST) 4 to 17 IU/L (7-35 units /ml)

SGPT (ALT) 3 to 15 IU/L (6-32 units /ml)

Liver Tissues are rich in SGPT.

In liver disease both transaminases are raised but SGPT shows much higher values.

Their determination is of extreme use in assessing the severity and prognosis of parenchymal liver diseases

It is the most sensitive diagnostic index.

The increase can even be such in the

prodromal stage, when jaundice has not

clinically appeared.

In infective hepatitis values as high as

1000 units have been observed.

SGPT IS Used as screening test in the outbreak of infective hepatitis viral hepatitis.

High values of SGPT are also obtained in, though the increase is comparatively less.

i. Toxic hepatitis – due to CCl4 poisoningII. Hepatitis due to drugs – Chlorpromazine

In obstructive Jaundice (extra hepatic) – values increase not

above 200 –300 IU/L.

Determination is effective in differential diagnosis of Jaundice.

SERUM ALKALINE PHOSPHATASE (3 –13 KAU/100 ml ; 23 –92 IU/L)

Found in a number of organs; Most plentiful in Bones and Liver.

Present also in small intestine, kidney placenta.

Used for many years in the differential diagnosis of Jaundice.

Raised levels are seen in both – infectious hepatitis (viral); Post hepatic Jaundice (extra hepatic obstruction).

In obstructive Jaundice raised ALK phosphatase level is much higher.

Values higher than 35 KA units are suggestive of obstructive Jaundice, where ALP rises up to 200 KAU.

35 KAU is the dividing line on the basis of which by the assay of ALP differential diagnosis between infectious hepatitis and obstructive Jaundice can be made.

Very high levels of ALP are also found in : -i) Xanthomatous biliary cirrhosis (in which

there is no extra-hepatic obstruction.ii) Space occupying lesions of liver.a) Abscessb) Primary carcinoma (Hepatoma)c) Metastatic carcinomad) Infiltrative lesion, Like lymphomae) Granuloma and amylodotis

ALP assays differentiateCholestatic Jaundice is Characterized by High ALP low amino Transferase.

Non-Cholestatic Jaundice converse occurs.

II . OTHER ENZYMES (not done routinely)

I. Serum 5' Nucleotidases ( 2 –17 IU/L) This enzyme hydrolyses nucleotides with a

phosphate group on carbon atom 5' of ribose.

Raised along with serum ALP in Liver disease.

Post hepatic jaundice raised up to 100 units. In bone diseases nucleotidases are normal

where as SALP is raised e.g. Paget’s diseases.

II. Serum Lactate Dehydrogenase (LDH) 70 – 240 IU/L

Increased activity is found particularly in infectious hepatitis.

The increase is not as great as the transaminase and its behaviour is less predictable as the enzyme increases in other diseases as well, like leukemia, pernicious anemia, megaloblastic and haemolytic anemia in renal diseases and generalized carcinosis.

III. Serum Iso-citrate Dehydrogenase (ICD) (Normal range 0.9 to 4.0 IU/L)

In liver diseases – A marked increase in ICD activity whether it is inflammatory like infectious hepatitis, malignancy or from drugs.

Large increase in infectious hepatitis – Serum activity normal on 3rd day.

In obstructive Jaundice – Normal value.Liver Cirrhosis – Serum activity normal or

slightly raised.

IV. Serum Cholinesterases (Normal value 2.17 to 5.17 IU/mL)

They are enzymes which hydrolyze esters of choline.

To give Choline and acetyl units

They are of two types1. True cholinesterases (present in nerve

tissue and RBC)

2.Pseudocholinesterases (present in liver, heart, muscles, intestine).

PseudocholinesterasesFormed in the Liver

Serum activity is reduced in liver cell damage and advanced cases of liver cirrhosis.

Normal activity in obstructive Jaundice

First enzyme of Urea cycle catalyzing

Ornithine + Carbonyl P Citrulline + PO4

Elevated markedly in viral hepatitis (10 – 200 fold), depending on the severity.

Slight elevation in Obstructive Jaundice.

V. Serum Ornithine Carbamyl Transferase (OCT) (8 – 20IU/L)

It is a Proteolytic enzyme which splits N – terminal residues from certain L – peptides.

Viral Hepatitis – mild to moderate increase – 30 –130 m IU.

Obstructive Jaundice – Marked increase, more in malignant obstruction

than benign.- Benign obstruction – 75 to 184 IU- Malignant obstruction – 67 to 340 IU

Marked rise in liver cell carcinoma.

VI. Serum Leucine amino peptidase (LAP 15 – 56 m IU

VII. Serum Hydroxy Butyrate Dehydrogenase (SHBD – 56 –125 IU/L)

• This enzyme acts on -OH Butyric Acid.

• Elevated levels in acute viral hepatitis.

VIII. Serum Glutamyl Transferase ( GI) Normal – 10 –47 IU/L

• Recently the importance of this enzyme in alcohol abuse has been stressed.

• The activity of this microsomal enzyme has been found to increase in heaptobilliary diseases, but unlike aminotransferases elevated levels do not necessarily indicate liver cells disruption, but may be due to enzyme induction by drugs such as phenobarbitons, phenytoin and alcohol

• Severe limitations have meant that this test now has only two practical uses.(a) An elevated GT implies, that elevated ALP is of hepatic origin(b) Useful in screening alcohol – Sudden increase in GT in chronic alcoholics suggest recent drinking bout of drinking alcohol.