MEDICAL USE OF ENZYMES

Name: Mohammad Reza Abdullahi

Matric No: G1629381
IIUM, Malaysia
Enzymes are used clinically in three principal way:
1) As indicators of enzyme activity or enzyme concentration in body fluids (e.g. serum and urine) in the
diagnosis and prognosis of various diseases.
2) As analytical reagents in the measurement of activity of other enzymes or nonenzyme substances
(e.g. substrates, proteins, and drugs) in body fluids
3) As therapeutic agents
Diagnosis and prognosis of disease
Enzyme assays employed in the diagnosis of disease are one of the most frequently used clinical laboratory
procedures. The most commonly used body fluid for this purpose is serum.
Enzymes in circulating plasma are either plasma-specific or non-plasma-specific. Plasma specific enzymes
are normally present in plasma, perform their primary function in blood, and have levels of activity that are
usually higher in plasma than in tissue cells. Examples are those enzymes involved in blood clotting (e.g,
thrombin), fibrinolysis (e.g. plasmin), and complement activation, as well as cholinesterase and
ceruloplasmin. These enzymes are synthesized mainly in the liver and released into the circulation at a rate
that maintains optimal steady-state concentration.
Non-plasma-specific enzymes are intracellular enzymes normally present in plasma at minimal levels or at
concentrations well below those in tissue cells. Their presence in plasma is normally due to turnover of tissue
cells, but they are released into the body fluids in excessive concentrations as a result of cellular damage or
impairment of membrane function. Tissue injury and impairment of membrane function can be caused by
diminished oxygen supply (e.g. myocardial infraction), infection (e.g. hepatitis), and toxic chemicals.
Proliferation of cells, with consequent increased turnover, can also raise levels in plasma of enzymes
characteristic of those cells (e.g. elevation of serum acid phosphatase in prostatic carcinoma).
Some important enzymes in various tissue are as follow:

Liver Enzymes
Enzymes in this category include 1) alanine aminotransferase, 2) aspartate aminotransferase, 3)
γ-gluta ltra sferase, alkali e phosphatase, a d ’-nucleotidase.

Clinicaly, the most common alterations in liver enzyme activities are 1) hepatocellular damage
ele ated a i otra sa i ase a ti ities a d holestasis ele ated alkali e phosphatase, ’-
u leotidase a d γ-glutamyltransferase activities.)

Aspartate Aminotransferase
Aspartate aminotransferase (AST) is an enzyme belonging to the class of transferases. It is
commonly referred to as a transaminase and is involved in transfer of an amino group between
aspartate a d α- keto acids. The older terminology , serum glutamic-oxaloacetic transaminase
(SGOT or GOT), may also be used. Pyridoxal phosphate functions as a coenzyme.
I oth a i otra sferases, p rido al ’ phosphate (P- ’-P) and its amino analogue,
pyridoxamine- ’- phosphate, function as coenzymes in amino transfer reactions. The P- ’-P is
bound to the apoenzyme and serves as a true prosthetic group. P- ’-P bound the apoenzyme
accepts the amino group from first substrate (aspartate or alanine) to form enzyme bound
pyridoxamine- ’- phosphate and the first reaction product, oxaloacetate or pyruvate,
respectively. The coenzyme in amino form then transfers its amino group to the second
substrate, 2-oxoglutarate, to form the second product, glutamate. P- ’-P is thus regenerated.

The transamination reaction is important in intermediary metabolism because of its function in

the synthesis and degradation of amino acids. The ketoacids formed by reaction are ultimately
oxidized by tricarboxylic acid cycle (TCA) to provide a source of energy.

Tissue source: AST is widely distributed in human tissue. The highest concentrations are found in
cardiac tissue, liver and skeletal muscle with smaller amounts found in the kidney, pancreas and
erythrocytes.

Clinical significance: Its evaluation mainly seen in hepatocellular disorder and skeletal muscle
involvement. In AMI, AST levels begins to rise within 6 to 8 hours, peak at 24 hours, and generally
return to normal within 5 days. However, because of the wide tissue distribution, AST levels are
not useful in the diagnosis of AMI.

AST elevations are frequently seen in pulmonary embolism and congestive heart failure. AST
levels are highest in acute hepatocellular disorders. In viral hepatitis, level may reach 100 times
ULN. In cirrhosis, only moderate levels (approximately 4 times ULN) are detected. Also, AST level
increase in skeletal muscle disorders, such as the muscular dystrophies, and inflammatory
conditions.

AST has two isoenzyme fractions located in the cell cytoplasm and mitochondria. The cytoplasmic
isoenzyme is the predominant form occurring in serum. In disorders producing cellular necrosis,
the mitochondrial form may be significantly increased. Isoenzyme analysis of AST is not routinely
performed in the clinical laboratory.
Alanine Aminotransferase
Alanine aminotransferase (ALT) is a transferase with enzymatic activity similar to that of AST.
Specifically, it catalyzes the transfer of an amino group from ala i e to α-ketoglutarate with the
formation of glutamate and pyruvate. The older terminology was serum glutamic-pyruvic
transaminase (SGPT or GPT).

Tissue source: ALT is distributed in many tissues, with comparatively high concentrations in the
liver. It is considered the more liver specific enzyme of the transferases.

Clinical significance: Clinical applications of ALT assay are confined mainly to evaluation of
hepatic disorders. Higher elevations are found in hepatocellular disorder than in extrahepatic or
intrahepatic obstructive disorders. In acute inflammatory conditions of the liver, ALT elevations
are frequently higher than those of AST and tend to remain elevated longer as a result of the
longer half-life of ALT in serum (16 and 24 hours, respectively).

Cardiac tissue contains a small amount of ALT activity, but the serum level usually remain normal
in AMI unless subsequent liver damage has occurred.

γ-Glutamyltransferase
peptidases catalyze the hydrolytic cleavage of peptides to form amino acid or smaller peptides
and so some individual enzymes act as amino acid transferases and catalyze the transfer of amino
a ids fro o e peptide to a other a i o a id or peptide. GTT atal zes the tra sfer of the γ-
gluta l group fro γ-glutamyl peptides and compounds to an acceptor such as amino acids,
ater a d other s all peptides. I ost iologi s ste , glutathio e ser es as the γ-glutamyl
donor.
The specific physiologic function of GGT has not been clearly established, but it is suggested that
GGT is involved in peptide and protein synthesis, regulation of tissue glutathione levels and the
transport of amino acids across cell membranes.

Tissue source: GGT activity is found primarily in tissue of the kidney, brain, prostate, pancreas
and liver. Clinical applications of assay, however, are confined mainly to evaluation of liver and
biliary system disorders.

Clinical significance: Increased GGT levels are not specific an are seen in most individual with
liver disease. GTT is elevated in hepatobiliary disorders so higher elevations are generally
observed in biliary tract obstruction. It increases in patients receiving enzyme-inducing drugs
such as warfarin, phenobarbital, and phenytoin (4 times ULN)

Because of the effects of alcohol on GGT activity, elevated GGT levels may indicate alcoholism,
particularly chronic alcoholism ( 2 to 3 times ULN). GTT levels are also elevated in other conditions
such as acute pancreatitis, diabetes mellitus and myocardial infraction.

Determination of GGT activity can be useful in the context of elevated ALP activity. GGT activity
is normal during pregnancy and in patients with bone disorders, conditions that exhibit elevated
ALP activity.

Alkaline phosphatase
ALP belongs to a group of enzymes that catalyze the hydrolysis of various phosphomonoesters at
an alkaline PH. It liberates inorganic phosphate from an organic phosphate ester with the
concomitant production of an alcohol. The optimal PH for reaction is 9.0 to 10.0, but optimal PH
varies with substrate used. The enzyme requires Mg2+ as an activator.
Tissue source: ALP activity is present on cell surface in most human tissues. The highest
concentrations are found in the intestine, liver, bone (i.e. osteoblasts), spleen, placenta and
kidney. Although the exact metabolic function of the enzyme is not yet understood, it appears
that ALP is associated with lipid transport in the intestine and with the calcification process in
bone. ALP occurs in several isoforms, some of which share a common primary structure. The liver
isoenzyme is the most abundant in adult serum, followed by the bone isoenzyme and a small
amount of ALP of intestinal origin.

Clinical significance: Increase in ALP activity are clinically significant in the investigation of
obstructive hepatobiliary disease and osteoblast medicated bone disease. ALP activity elevations
of 3 to 10 times the upper limit of the reference interval is commonly observed in biliary tract
obstruction. However, a single increase in the ALP level is not considered diagnostic.

ALP activity elevations related to bone disease are common in osteoblast related disorder,
including Paget disease (osteitis deformans), osteomalacia, and rickets.

A transient physiologic increase in the serum ALP concentration is observed in infants and
children and is attributed to normal bone growth. ALP activity elevations can be detected in
serum of pregnant women starting at 16 eeks’ gestatio .

5’-Nucleotidase
’- e leotidase NTP is a phosphatase that a ts o l o u leoside ’-phasphates, such as AMP
and adenylic acid, releasing inorganic phosphate.

NTP is a glycoprotein that is widely distributed throughout the tissues of the body and is
principally localized in the cytoplasmic membrane of the cells in which it occurs. Despite its
ubiquitous distribution, serum NTP activities appear to reflect hepatobiliary disease with
considerable specificity.

The assay of NTP activity has been considered of value as an addition to measurement of
nonspecific total ALP in patients with suspected hepatobiliary disease and abnormal NTP activity
is routinely interpreted as evidence of hepatic origin of increased ALP activity in serum. However,
approximately half of individuals in whom liver ALP activity is increased in serum may
simultaneously show a normal NTP activity. Alternatively, increased NTP activity in the serum of
patients with normal liver ALP activity is very often associated with the presence of liver disease.

Pancreatic Enzymes
The most commonly serum biomarkers for investigation of pancreatic disease, and more
specifically acute pancreatitis are digestive enzymes (P-type) amylase and lipase.

Amylase
Amylase (AMS) is an enzyme belonging to the class of hydrolases that catalyze the breakdown of
star h a d gl oge . α-a lase atta ks o l the α, -4 glycosidic bonds to produce degradation
products consisting of glucose, maltose and intermediate chains called de tri s hi h o tai α,
1-6 branching linkages. AMS requires calcium and chloride ions for its activation.

Tissue source: The acinar cells of the pancreas and the salivary glands are the major tissue
sources of serum AMS. Lesser concentrations are found in skeletal muscle and the small intestine
and fallopian tubes. AMS is the smallest enzyme so it is readily filtered by the renal glomerulus
and also appears in the urine.

The salivary isoform of amylase (s-AMS) initiates starch digestion in the mouth and esophagus
and becomes inactivated by the acidic PH in the stomach. The pancreatic isoenzyme of amylase
(p-AMS) continues the hydrolysis of polysaccharides after the stomach contents reach the small
intestine, where p-AMS is secreted from the pancreas.

Clinical significance: Total blood AMY activity is physiologically low and constant and is greatly
increased in cases of acute pancreatitis and salivary gland inflammation. During the progression
of acute pancreatitis, transient increase in amylase activity are observed 5 to 8 hours after the
onset of symptoms, with a peak seen 12 hours and a return to normal after 3 to 4 days. Because
elevated amylase activity is observed in other disorders with similar symptoms, it is considered a
nonspecific test when used alone. Additional tests, including urinary amylase and lipase activity
determinations, increase its diagnostic specificity. Other pathologies with elevated amylase
activity levels include renal insufficiency, biliary tract disorders, ectopic pregnancy and diabetic
ketoacidosis.

Lipase
Lipase catalyzes the hydrolysis of triglycerides to produce glycerol and fatty acids. The enzymatic
activity of human lipase is specific for the ester bonds at position 1 and 3, it generates a 2-
acylglycerol and release fatty acids. The reaction rate is accelerated by the presence of bile salts
and the coenzyme colipase.
Tissue source: LPS concentration is found primarily in the pancreas, which has concentrations up
to 5000 times higher than in other tissues.

Clinical significance: Clinical assays of serum LPS are confined almost exclusively to the diagnosis
of acute pancreatitis. Lipase activity in the sera of non-diseased individuals is low. Any increase
of lipase activity determinations is used to diagnose acute pancreatitis. During an acute
pancreatitis episode, lipase activity increases are observed 5 to 8 hours after the onset of
symptoms, with a peak at 24 hours and a return to normal after 8 to 14 days.

Increased serum lipase levels are seen in other acute disorders with intraabdominal involvement,
although less frequently than the nonspecific serum amylase elevations. Serum elevations of
lipase activity are more specific than increase in amylase activity for the diagnosis of acute
pancreatitis.
 Muscle enzymes
Enzymes in this category contain creatine kinase and aldolase.

Creatine Kinase

Creatine kinase (CK) catalyze the reversible phosphorylation of creatine (cr) to creatine
phosphate by adenosine triphosphate (ATP)

The forward reaction occurs in mitochondria, in which CK catalyzes the conversion of ATP to ADP
and stores energy as creatine phosphate. In the cytoplasm, CK phosphorylates ADP to produce
the high-energy ATP that is necessary for muscle to contract.

Tissue source: CK is widely distributed in tissue, with highest activities found in skeletal muscle,
heart muscle, and brain tissue. CK is present in much smaller quantities in other tissue sources,
including the bladder, placenta, gastrointestinal tract, thyroid, uterus, kidney, lung, prostate,
spleen, liver and pancreas.

The CK molecule is composed of two subunits. M and B. different subunit combinations generate
three major isoenzymes: CK-BB (CK1), CK-MB (CK2) and CK-MM (CK3). CK-BB is found primarily
in brain and intestines, and it is found in extremely low concentrations in serum. CK-MB is found
in small amounts in skeletal muscle and is abundant in heart muscle. It represents 2% to 3% of
the total CK activity in serum. CK-MM is the most abundant isoenzyme, its activity is distributed
primarily in skeletal and heart muscles. Between 97% to 98% of the total CK activity in serum is
attributed to CK-MM.

Clinical significance: because of its high concentration in skeletal muscle, total CK activity in non-
diseased individuals varies depending on age, gender, race, percentage of muscle mass and
physical condition.

CK levels are frequently elevated in disorders of cardiac and skeletal muscle. The CK level is
considered a sensitive indicator of acute myocardial infraction (AMI) and muscular dystrophy. CK
elevations are commonly seen when direct muscle injury occurs, such as after intense physical
activity, a sever fall or surgery.

Values for the MB isoenzyme range from undetectable to trace ( 6% of total CK). Injury to both
cardiac and skeletal muscle account for the majority of cases of CK-MM elevations.

Following MI, the CK-MB levels begin to rise within 4 to 8 hours, peak at 12 to 24 hours and return
to normal levels within 48 to 72 hours. The specificity of CK-MB levels in the diagnosis of AMI can
be increased if interpreted in conjunction with LDH isoenzymes and troponins and if measured
sequentially over a 48 hour period to detect the typical rise and fall of enzyme activity seen in
AMI.

Non enzyme proteins (troponin I and troponin T) have been used as a more sensitive and specific
marker of myocardial damage. These proteins are released into the bloodstream earlier and
persist longer than CK and its isoenzyme CK-MB.

Aldolase

Aldolase (ALD) catalyzes the splitting of d-fructose 1,6 diphosphate to d-glyceraldehyde 3

phosphate( GLAP) and dihydroxyacetone phosphate (DAP) which is an important reaction in the
glycolytic breakdown of glucose to lactate.

Serum ALD determinations have been of some clinical interest in diseases of skeletal muscle.
Some researchers believe that increased ALD activity in combination with the CK/AST ratio is
useful in distinguishing neuromuscular atrophies from myopathies. In general, however,
measurement of ALD activity in the serum of subjects with suspected muscle disease does not
add information to that available more readily from measurement of other enzymes, especially
CK.

Lactate Dehydrogenase

LD is an enzyme that catalyzes the interconversion of lactic to pyruvic acids. It is a hydrogen-

transfer enzyme that uses the coenzyme NAD+.
Tissue source: LD is widely distributed in the body. High activities are found in the heart, liver,
skeletal muscle, kidney and erythrocyte. Lesser amounts are found in the lung, smooth muscle
and brain.

Clinical significance: increased levels are found in cardiac, hepatic, skeletal muscle and renal
disease as well as in several hematologic and neoplastic disorders. The highest levels of total LDH
are seen in pernicious anemia and hemolytic disorders. Liver disorders, such as viral hepatitis and
cirrhosis show slight elevations of two to three times ULN. In AMI, LDH levels begin to rise within
12 to 24 hours, reach peak levels within 48 to 72 hours, and may remain elevated for 10 days.
Skeletal muscle disorders and some leukemias contribute to increased LDH levels. The enzyme
can be separated into five major fractions, each comprising four peptide chains of two types M
and H. In the sera of healthy individuals, the major isoenzyme fraction is LDH-2, followed by LDH-
1, LDH-3, LDH-4 and LDH-5. LDH-1 and LDH-2 are present to approximately the same extent in
the tissues. However, cardiac tissue and red blood cells contain a higher concentration of LDH-1.
Therefore, in conditions involving cardiac necrosis (AMI) and intravascular hemolysis, the serum
levels of LDH-1 will increase to a point at which they are present in greater concentration than
LDH-2, resulting in a condition known as the LDH flipped pattern (LDH- LDH-2). This flipped
pattern is suggestive of AMI. However, LDH is not specific to cardiac tissue and is not a preferred
marker of diagnosis of AMI.

Glycogen phosphorylase

Glycogen phosphorylase uses inorganic HPO42+ to split glucose from the polysaccharide chains of

glycogen.

The glucose 1-phosphate so formed can be used for ATP synthesis in muscle or converted to free
glucose in the liver. Pyridoxal phosphate is an essential cofactor in the glycogen phosphorylase
reaction. It has two form, glycogen phosphorylase a (phosphorylated) which is active form and
phosphorylase b (unphosphorylated) which is less active.

Lack of glycogen phosphorylase result in Mc Ardle disease (glycogen storage disease type 5)
which causes muscle cramp and muscle damage due to inadequate energy supply.

Mutations in the muscle isoform of glycogen phosphorylase (PYGM) are associated with
McArdle's Disease (glycogen storage disease type V). More than 65 mutations in the PYGM gene
that lead to McArdle disease have been identified to date. Symptoms of McArdle disease include
muscle weakness, myalgia, and lack of endurance, all stemming from low glucose levels in muscle
tissue.
Mutations in the liver isoform of glycogen phosphorylase (PYGL) are associated with Hers'
Disease (glycogen storage disease type VI). Hers' disease is often associated with mild symptoms
normally limited to hypoglycemia, and is sometimes difficult to diagnose due to residual enzyme
activity.

The brain isoform of glycogen phosphorylase (PYGB) has been proposed as a biomarker for
gastric cancer.

Enzymes as analytical reagents

The use of enzymes as analytical reagents in the clinical laboratory has found widespread
application in the measurement of substrates, drugs and activity of other enzymes. Enzyme-
dependent procedures, used in the assay of substrates such as glucose, urea, uric acid, and
cholesterol, provide many dvantages over classical chemical procedures. These advantages
include specificity of the substrate that is being measured and direct measurement of the
substrate in a complex mixture that avoids preliminary separation and purification steps such as
serum. A reagent enzyme can be used in substrate measurements by two methods: end-point
and rate assays. In an end-point assay, the substrate is completely converted to product before
it is measured; in the rate assay, a change in substrate concentration produced during a fixed-
time interval is measured. This second method, also referred to as the two-point kinetic method,
when carried out under constant conditions of pH, temperature, amount of enzyme, and time
interval, yields very accurate values for substrate concentration.
Enzymes as therapeutic agents
Enzymes have found a few applications as therapeutic agents. Some examples are transfusion of
fresh blood or its active components in bleeding disorders, oral administration of digestive
enzymes in digestive diseases, administration of fibrinolytic enzymes (e.g. streptokinase) to
recanalize blood vessels occluded by blood clots (thrombi) in thromboembolic disorders (e.g.
pulmonary embolism, acute myocardial infraction), treatment of selected disorders of inborn
errors of eta olis e.g. Gau her’s disease , a d a er therap e.g. L-asparaginase in acute
lymphocytic leukemia).

Some important therapeutic enzymes are listed at the following table.