Enzymes pdf - د. هدف

Clinically
Important

Enzymes and
Diagnostic
Applications

(part I)

Prof. Dr. H.D.El-Yassin

October 2013

Prof. Dr. H.D.El-Yassin

2013

Objectives

1. To list some clinically important enzymes

2. To state the distribution and applications of clinically important enzymes

3. to interpret the biochemical results of serum enzymes for three cases of different

liver diseases

4. To illustrate the serum enzyme patterns in different diseases

5. To assess the serum enzyme patterns in viral hepatitis

Clinically Important Enzymes and

Diagnostic Applications

Distribution and application of clinically important enzymes

Enzyme

Principle Sources of

Enzyme in blood

Clinical applications

Alanine

aminotransferase

Liver

Hepatic parenchymal diseases

Alkaline

phosphetase

Liver, bone, intestinal

mucosa, placenta

Bone diseases, hepatobiliry diseases

Amylase

Salivary glands, pancreae

Pancreatic diseases

Aspartate

aminotrasferase

Liver, skeletal muscle,

heart erythrocytes

Hepatic parenchymal disease, muscle

disease

Cholinesterase

Liver

Organophosphorus insecticide poisoning,

hepatic parenchymal disease

Creatine kinase

Skeletal muscle, heart

Muscle diseases(M.I.)

γ-glutamyl

transferase

Liver

Hepatobiliary diseases, marker of alcohol

abuse

Lactate

dehydrogenase

Heart, liver, skeletal

muscle, erythroctes,

platelets, lymph nodes

Hemolysis, hepatic parenchymal diseases,

tumor marker

lipase

Pancreas

Pancreatic diseases

5'-nucleotidase

Liver

Hepatbiliary diseases

Trypsin

pancreas

Pancreatic diseases

Prof. Dr. H.D.El-Yassin

2013

Pancreatic enzymes:

Acute pancreatitis is an inflammatory process in which pancreatic enzymes are

activated and cause autodigestion of the gland. It is a result of anatomical changes that

arise from two events.

1. The first is the autodigestion of the acinar cells by inappropriate activation of the

pancreatic enzymes (especially trypsinogen) within the cell.

2. The second is the cellular injury response that is mediated by proinflammatory

cytokines.

There are some enzymes that are synthesized and stored as the active enzymes in the

zymogen granules. These include

α-amylase, lipase, colipase, RNase, and DNase.

α-Amylase: (EC3.2.1.1; 1,4- α-D-glucan glucanohydrolase; AMY) is an

enzyme of the hydrolyase class that catalyzes the hydrolysis of 1,4-

α-glycosidic

linkages in polysaccharides. AMYs are calcium metaloenzymes, with the

calcium absolutely required for functional integrity.

AMYs normally occurring in human plasma are small molecules with molecular

weights varying from 54 to 62 kDa. The enzyme is thus small enough to pass the

glumeruli of the kidneys and AMY is the only plasma enzyme physiologically found in

urine. The AMY activity present in normal serum and urine is of pancreatic (P-AMY)

and salivary gland (S-AMY) origin.

Clinical Significance

Normal values of amylase: 28-100 U/L =0.48-1.7 µkat/L

CAUSES OF RAISED PLASMA AMYLASE ACTIVITY



Marked increase (five to 10 times the upper reference limit):

o acute pancreatitis:

o severe glomerular impairment:

o perforated peptic ulcer especially if there is perforation into the lesser

sac.

Pancreatic pseudocyst. If the plasma amylase activity fails to fall after an attack of

acute pancreatitis there may be leakage of pancreatic fluid into the lesser sac (a

pancreatic pseudocyst). Urinary amylase levels are high, differentiating it from

macroamylasaemia. This is one of the few indications for estimating urinary amylase

activity, which is inappropriately low relative to the plasma activity if there is glomerular

impairment or macroamylasaemia

Prof. Dr. H.D.El-Yassin

2013

Macroamylasaemia. In some patients a high plasma amylase activity is due to a low

renal excretion of the enzyme, despite normal glomerular function. The condition is

symptomless; it is thought that either the enzyme is bound to a high molecular

weight plasma component such as protein, or that the amylase molecules form large

polymers that cannot pass through the glomerular membrane. This harmless

condition may be confused with other causes of hyperamylasaemia

2. Lipase: (EC 3.1.1.3; triacylglycerol acylhydrolase; LPS).is a single

–

chain glycoprotein with molecular weight of 48 kDa. For full catalytic

activity and greatest specificity the presence of bile salts and a cofactor

called colipase, which is secreted by the pancreas, is required. LPS is a

small molecule and is filtered through the glomerulus. It is totally

reabsorbed by the renal tubules, and it is not normally detected in urine.

Clinical Significance

Normal values: 40-200 U/L

Plasma lipase levels are elevated in acute pancreatitis and carcinoma of the

pancreas.

Note: serum amylase is increased in mumps, pancreatic disease or due to some other

cause, where as lipase is increased only in pancreatitis. Therefore, the determination of

both amylase and lipase together helps in the diagnosis of acute pancreatitis.

Prof. Dr. H.D.El-Yassin

2013

Liver enzymes:

The assay of serum enzymes is very useful for the differential diagnosis and monitoring

of various heptobiliy disorders.

There are three types of enzymes:

1. Enzymes which are normally present inside the hepatocytes released into the

blood when there is a hepatocellular damage= markers of hepatocellular

damage.

2. Enzymes which are primary membrane bound (plasma membrane or side of

hepatocytes) = markers of cholestasis.

3. Enzymes which are synthesized in the hepatocyte= indicates disturbances in

the hepatocellular synthesis.

1.Markers of hepatocellular damage.

1. Aminotransaminases

The transaminases are enzymes involved in the transfer of an amino group from a 2-

amino- to a 2-oxoacid: they need the cofactor, pyridoxal phosphate for optimal activity.

They are widely distributed in the body.

The 2-oxoglutarate/L-glutamate couple serves as one amino group acceptor and donor

pair in all amino-transfer reactions; the specificity of the individual enzymes derives from

the particular amino acid that serves as the other donor of an amino group. Thus AST

catalyzes the reaction:

Prof. Dr. H.D.El-Yassin

2013

ALT catalyzes the analogous reaction:

The reactions are reversible, but the equilibrium of AST and ALT reactions favor formation

of aspartate and alanine respectively.

o Location:

o AST present in cytosol and mitochondria

o ALT located in cytosol of liver

o In the liver, the concentration of ALT per unit weight of the tissue is more than AST.

o These enzymes are more important in assessing and monitoring the degree of liver

cell inflammation and necrosis.

o The highest activities of ALT are found in hepatocytes and muscle cells.

o Hepatocytes have very high activity of ALT, therefore elevations in serum ALT are

considered to be relatively specific for liver disease.

o AST may be elevated in other forms of tissue damage, such as myocardial

infarction, muscle necrosis and renal disorders.

o In liver disease, the ALT level is increased markedly compared to AST.

o In acute viral hepatitis there is a 100-1000 times increase in both ALT and AST but

ALT level is increased more than that of AST

Prof. Dr. H.D.El-Yassin

2013

Table: Aminotransferase activities in human tissues, relative to serum as unity

a. Aspartate Transaminase (EC 2.6.1.1; L-aspartate:2-oxoglutarate

aminotransferase; AST)

Clinical Significance

Normal values of AST:

male:

<35 U/L = <0.60 µkat/L

Female:

<31 U/L = <0.53 µkat/L

AST (glutamate oxaloacetate transaminase. GOT) is present in high concentrations in

cells of cardiac and skeletal muscle, liver, kidney and erythrocytes. Damage to any of

these tissues may increase plasma AST levels. Half- life = 17 hours.

CAUSES OF RAISED PLASMA AST ACTIVITIES

• Artefactual.

Due to in vitro release from erythrocytes if there is haemolysis or if separation of

plasma from cells is delayed.

• Physiological.

During the neonatal period (about 1.5 times the upper adult reference limit).

• Marked increase (10 to 100 times the upper adult reference limit):

Circulatory failure with 'shock' and hypoxia:

Myocardial infarction

Acute viral or toxic hepatitis.

Prof. Dr. H.D.El-Yassin

2013

b. Alanine Transaminase (EC 2.6.1.2; L-alanine:2-oxoglutarate

aminotransferase; ALT)

Clinical Significance

Normal values of ALT:

male:

<45 U/L = <0.77 µkat/L

Female:

<34 U/L = <0.58 µkat/L

ALT (glutamate pyruvate transaminase. GPT) is present in high concentrations in liver

and to a lesser extent, in skeletal muscle, kidney and heart. Half- life = 47 hours

In liver damage, both enzymes are increased but ALT increases more. In myocardial

infarction AST is increased with little or no increase in ALT.

CAUSES OF RAISED PLASMA ALT ACTIVITIES



Marked increase (10 to 100 times the upper limit of the adult reference range

circulatory failure with 'shock' and hypoxia:

Acute viral or toxic hepatitis.



Moderate increase:

Cirrhosis (may be normal or up to twice the upper adult reference limit): infectious

mononucleosis (due to liver involvement):

Liver congestion secondary to congestive cardiac failure:

cholestatic jaundice (up to 10 times the upper reference limit in adults); surgery or

extensive trauma and skeletal muscle disease (much less affected than AST)

same AST except
Myocardial infarction

Prof. Dr. H.D.El-Yassin

2013

2.Markers of cholestasis

I. Alkaline phosphatase (EC 3.1.3.1; orthophosphoric-monoester

phosphohydrolase [alkaline optimum]; ALP). Half-life= 10 days

Clinical Significance

The alkaline phosphatases are a group of enzymes that hydrolyse organic phosphates

at high pH. They are present in most tissues but are in particularly high concentration in

the osteoblasts of bone and the cells of the hepatobiliary tract, intestinal wall, renal

tubules and placenta. The exact metabolic function of ALP is unknown but it is probably

important for calcification of bone.

In adults plasma ALP is derived mainly from bone and liver in approximately equal

proportions: the proportion due to the bone fraction is increased when there is

increased osteoblastic activity that may be physiological

Causes of raised Plasma ALP activity



Physiological:

There is a gradual increase in the proportion of liver ALP with age:

in the elderly the plasma bone isoenzyme activity may increase slightly.



Bone disease

rickets and osteomalacia

secondary hyperparathyroidism .



Liver disease-.



Malignancy.

bone or liver involvement or direct tumor production.

POSSIBLE CAUSES OF LOW PLASMA ALP ACTIVITY

• Arrested bone growth

• Hypophosphatasia: an autosomal recessive disorder, associated with rickets or

osteomalacia.

Table: reference intervals for alkaline phosphatase activities inn serum

ISOENZYMES OF ALKALINE PHOSPHATASE

Prof. Dr. H.D.El-Yassin

2013

Bone disease with increased osteoblastic activity, or liver disease with involvement of the

biliary tracts, are the commonest causes of an increased total alkaline phosphatase

activity.

Assays for ALP isoenzymes are needed when:

I. The source of an elevated ALP in serum is not obvious and should be

clarified.

II. The main clinical question is concerned with detecting the presence of

liver or bone involvement

III. In the case of metabolic bone disorders, to ascertain any modifications in

the activity of osteblastes to monitor the disease activity and the effect of

appropriate therapies.

2. Gamma-glutamyl-transferase

(EC 2.3.2.21; γ-glutamyl-peptide: amino

acid γ -glutamyletransferase; GGT): catalyzes the transfere of the γ –

glutamyl group from peptides and compounds that contain it to an acceptor

Gamma-glutamyltransferase occurs mainly in the cells of liver, kidneys, pancreas

and prostate. Plasma GGT activity is higher in men than in women.

Clinical Significance

Normal values for GGT

male:

<55 U/L = <0.94 µkat/L

Female:

<38 U/L = <0.65 µkat/L

Causes of raised plasma GGT activity

• Induction of enzyme synthesis, without cell damage, by drugs or alcohol.

• Hepatocellular damage, such as that due to infectious hepatitis:

A patient should never be labeled an alcoholic because of a high plasma GGT activity

alone.

Other enzymes

Glutamate dehydrogenase (EC 1.4.1.3; L-glutamate: NAD(P)

oxidoreductase,

deaminating; GLD) is a mitochondrial enzyme found mainly in the:

a. liver

b. heart muscle

c. kideys

but small amounts occur in other tissue, including

d. brain

e. skeletal muscle tissue

f. leukocytes

Prof. Dr. H.D.El-Yassin

2013

Clinical significance

GLD is increased in serum of patients with hepatocellular damage offering differential

diagnostic potential in the investigation of liver disease, particularly when interpreted in

conjunction with other enzyme test results. The key to this differential diagnostic

potential is to be found in the intraorgan and intracellular distribution of the enzyme. As

an exclusively mitochondrial enzyme, GLD is released from necrotic cells and is of

value in estimation of the severity of liver cell damage. GLD activity in serum is stable at

4ºC for 48 hours and at -20ºC for several weeks. The GLD upper reference limits are

6U/L (women) and 8U/L (men), when a method optimized at 37ºC is used.

Clinical Cases:
Case1:

A healthy 43- year old male, on no medication, had the following

results of tests performed during a private healthcare screening program.
Plasma
Bilirubin 43µ mol/L (< 20)
Unconjucated bilirubin 36µ mol/L (< 5)
Alanine aminotransferase 21U/L (< 42)
Alakaline phosphatase 126 U/L (< 250)
Albumin 40 g/L (35-45)
Gamma-glutamyltransferase 42 U/L (< 55)
Urinary bilirubin negative
Normal full blood count, reticulocytes and blood film.
Discussion

The raised concentration of the plasma bilirubin is predominant ly unconjugated. There

is no evidence of haemolysis and the other liver function tests are normal. A likely

diagnosis is of Gilbert's syndrome. This is a common condition and its diagnosis is

based on the exclusion of liver disease and heamolisis in the presence of a modest

concentration of unconjugated hyper bilirubineamia.

Case2 :

A 50-year old known alcoholic make attended the general medical

clinic because of ascites and the following abnormal liver test results
Plasma:
Bilirubin 52µ mol/L (< 20)
Alanine amino transferase 76 U/L (< 42)
Alakaline phosphatase 271 U/L (< 250)
Albumin 18 g/L (35-45)
Gamma-glutamyltransferase 324 U/L (< 55)
Urinary bilirubin and protin normal.
Discussion

The abnormal liver test and hypoalbuminaemia together with ascites supported the

diagnosis of cirrhosis, secondary to his alcohol problem. Hypoalbumineamia may be

due to many disorders, such as gross protienuria, but in the presence of hepatic

disease suggests a reduction in hepatic synthetic typical of cirrhosis.

Prof. Dr. H.D.El-Yassin

2013

Serum Patterns in different liver diseases

Ref: Schmidt E, Schmidt FW.“Brief guide to practical enzyme diagnosis” , Houston,
1977, Boehringer Mann-heim Diagnostics.

Prof. Dr. H.D.El-Yassin

2013