Clinical enzymology for first

year medical students

biochemistry

1
Prepared by:
Fatima abdelmalik
2 Units of enzyme activity
–The enzyme unit, or international unit for enzyme (U or IU) is
a unit of enzyme's catalytic activity.

–1 U (μmol/min) is defined as the amount of the enzyme that

catalyzes the conversion of one micromole of substrate per
minute under the specified conditions of the assay method.

–The specified conditions will usually be the optimum

conditions, which including temperature, pH and substrate
concentration, that yield the maximal substrate conversion
rate for that particular enzyme.
3 Enzymes Application In Medicine
–Diagnostic indicators – the activities of many enzymes are
routinely determined in plasma for diagnostic purposes in
diseases of the heart, liver, skeletal muscle, pancreas and
other tissues.

– Diagnostic tools – use as chemicals in clinical laboratory

assays

–Therapeutic agents – several enzymes are used as drugs;

enzymotherapy
4 Enzymes In Clinical Diagnosis
Enzymes: secretory: produced by tissues (namely liver) to
act in plasma as: prothrombin, plasminogen,
cerruloplasmin, choline esterase, lipoprotein lipase

intracellular : function intracellulary, have no

physiological use in plasma
- membrane bound ALP.
- cytosolic – ALT, AST.
- mitochondrial – AST.
- lysosomal - ACP
5
–Healthy individuals - levels of intracellular enzymes fairly
constant: the rate of enzyme release from cells into
plasma balanced by the rate of removal of enzyme
protein from plasma

–Elevated enzyme activity in the plasma – reflect tissue

damage accompanied by increased release of
intracellular enzyme

–Many diagnostically important enzymes = isoenzymes –

pattern of isoenzymes in plasma (determined
electroforetically)
 Examples of enzymes commonly assayed for diagnostic purposes
6
Enzyme Location Cause of elevated plasma level

Acid phosphatase - ACP Prostate Prostatic cancer

Alkaline phosphatase – ALP Bone, liver Rickets, hypoparathyroidism,

osteomalacia, obstructive
jaundice, cancer of bone/liver

Alanine aminotransferase – ALT Liver (muscle, Hepatitis, jaundice, circulatory

heart, kidney) faillure with liver congestion

Aspartate aminotransferase – AST Heart, muscle, Myocardial infarction, muscle

red cells, liver damage, anemia, hepatitis,
circulatory faillure with liver
congestion

Amylase - AM Pancres Acute pancreatitis, peptic ulcer

γ-Glutamyl transferase – GMT Liver, kidney, Hepatitis, alcoholic liver

pancreas damage, cholestasis
 Enzyme Location Cause of elevated plasma level
7
Creatine kinase – CK
CK-MB Heart Myocardial infarction
CK-MM Skeletal muscle Muscular dystrophy

Lactate dehydrogenase – LD
LD1 > LD2 Heart, kidney, Myocardial infarction, kidney
blood cells disease, megaloblastic anemia,
leukemia
LD2, LD3 Leukemia
LD5 Liver, muscle Liver disease, muscle damage
8 Enzymes In Therapy
– Orally administered enzymes: treatment of a variety disorders
- digestive, gastrointestinal, pancreatic:Mixtures of enzymes of
plant and/or animal origin include proteinases, amylase, lipase

– Injected enzymes: treatment of myocardial infarction (MI) and

pulmonary embolism by thrombolytic enzymes as Streptokinase
and Urokinase .
- viral diseases (herpes, AIDS) treated by azidothymidine(AZT)
- cancer :asparaginase, glutaminase in treatment of acute
lymphoblastic leukemia
9
Enzymes Use In Laboratory Assays
– Enzymes make excellent analytical reagents due to their specificity, selectivity
and efficiency than chemical methods . They are often used to determine the
concentration of their substrates (as analytes) in the blood, plasma/serum and
urine

– Components of commercial kits or diagnostic strips

- determination of glucose - glucose oxidase, peroxidase
cholesterol - cholesterol esterase, cholesterol oxidase
peroxidase,
urea – urease, ……. in blood, plasma, serum

– Markes in the immunochemical analysis

- ELISA (=enzyme-linked immunoadsorbent assay) – peroxidase, alkaline
phosphatase
10 Classification of enzymes according to function
–enzymes are divided into two types :
* Functional plasma enzymes.
* Non-functional plasma enzymes.

– 1)Functional plasma enzymes: Certain enzymes,

proenzymes, and their substrates are present at all times
in the circulation of normal individuals and perform a
physiologic function in the blood.
– Examples of functional plasma enzymes: Lipoprotein
lipase, Pseudo cholinesterase, Widely secreted from liver .
11 –Nonfunctional plasma enzymes : numerous enzymes that
perform no known physiologic function in blood.
–These apparently nonfunctional plasma enzymes arise
from the routine normal destruction of cells.
–Measurement of non functional enzymes is important for:
–Diagnosis of diseases: Tissue damage or necrosis resulting
from injury or disease is generally accompanied by
increases in the levels of several nonfunctional plasma
enzymes

–Prognosis of the disease: follow up the effect of treatment

by measure plasma enzyme before and after treatment.
 Source of Non functional plasma enzyme
12
–Cell damage with the release of its content of enzymes
into blood e.g. Myocardial infarction and viral hepatitis

– Obstruction of normal pathways e.g. Obstruction of bile

duct increases alkaline phosphatase

– Increase of the enzyme synthesis e.g. bilirubin increases

the rate of synthesis of alkaline phosphatase in
obstructive liver disease

– Increased permeability of cell membrane as in hypoxia

 Abnormal enzyme level
13
– causes of increase serum level of enzymes:
–increased cell turnover
–cellular proliferation (e.g. neoplasia)
–increased enzyme synthesis (enzyme induction)
–obstruction to secretion
–decreased clearance.
–Causes of decreased serum level of enzymes:
– inhibition of the activity by drugs
–inhibition of the synthesis of enzymes due to cell damage
or drugs
14
Enzymes used in diagnosis of liver disorders
– Serum enzyme tests can be grouped into two categories:
– Enzymes whose elevation in serum reflects damage to hepatocytes:
Aminotransferases (transaminases) are sensitive indicators of liver cell
injury and are most helpful in recognizing acute hepatocellular
diseases such as hepatitis. These include:
1) Aspartate aminotransferase (AST).
2) Alanine aminotransferase (ALT).

– Enzymes whose elevation in serum reflects cholestasis:

1)Alkaline phosphatase
2) 5'-nucleotidase.
3) γ-Glutamyl transpeptidase (GGT).
15 Amino Transferases
– The Aminotransferases are normally present in the serum in low
concentrations. These enzymes are released into the blood in
greater amounts when there is damage to the liver cell membrane
resulting in increased permeability
– Aspartate Amino Transferase (AST): It is also called as serum
glutamate-oxaloacetate transaminase (SGOT)
16
Aspartate Amino Transferase (AST):
–Normal serum level of AST is 8-40 U/L.

–AST is found in the liver, cardiac muscle, skeletal muscle,

kidneys, brain, pancreas, lungs, leukocytes, and
erythrocytes in decreasing order of concentration

–It is significantly elevated in myocardial infarction. It if

moderately elevated in liver diseases.
17
– Alanine Amino Transferase (ALT):It is also called as serum
glutamate-pyruvate transaminase (SGPT)

– Normal serum level of ALT is 5-30 U/L

– Very high values (100 to 1000 U/L) are seen in acute hepatitis, either
toxic or viral in origin.
18 – Both ALT and AST are increased in liver diseases, but ALT >AST.

– Moderate increase (25 to 100 U/L) may be seen in chronic liver

disease such as cirrhosis, and malignancy in liver.

– A sudden fall in ALT level in cases of hepatitis is a very bad

prognostic sign.

– In Acute Alcoholic Hepatitis The AST level is higher than the ALT level,
and the ratio is greater than 2:1 in 70% of patients. A ratio greater
than 3 is strongly indicative of alcoholic hepatitis
19 Alkaline phosphatase(ALP)

–Present in liver also found in bone and in smaller quantities

in the intestines, kidneys, and white blood cells.

–Normal serum level of ALP is 40-125 U/L

–Increase in cholestatic liver disease (e.g. common bile

duct obstruction, intrahepatic duct obstruction [including
by metastases], primary biliary cholangitis, primary
sclerosing cholangitis, and drug-induced cholestasis)
 Alkaline Phosphatase (ALP)
20

–Blood levels of alkaline phosphatase also increase

two to four times during pregnancy. This is a result of
additional alkaline phosphatase produced by the
placenta

–Placental ALP, elevates in a variety of malignancies,

including ovarian, lung, gastrointestinal cancers and
Hodgkin’s disease.
21 Gamma-glutamyltransferase (GGT)
–Present in the liver and also present in the intestines,
kidneys, pancreas, and prostate, but not in bone.

–Normal serum value of GGT is 6-45 U/L in male and 5-30

U/L in female.

–can be useful in determining whether elevated ALP is of

bone or liver origin.
22 NUCLEOTIDE PHOSPHATASE (NTP)
–It is also known as 5' nucleotidase.
–It is a marker enzyme for plasma membranes.
–Normal NTP level in serum is 2-10 U/L.
– It is moderately increased in hepatitis and highly elevated
in biliary obstruction.
– Unlike ALP, the level is unrelated with osteoblastic activity
and therefore is unaffected by bone diseases.
23 Diagnosis of Acute Myocardial Infarction (AMI)
– diagnosis of myocardial infarction is created by integrating
the history of the presenting illness and physical
examination with electrocardiogram(ECG) findings and
cardiac markers.

–patient is diagnosed with myocardial infarction if two

(probable) or three (definite) of the following criteria are
satisfied:
1) Clinical history of ischemic type chest pain lasting for
more than 20 minutes, minority of patients may have a silent
MI as in patients with DM.
24 2) Changes in serial ECG tracings, some of patients (30%)
may present with equivocal or absent ECG changes.

3) Rise of serum cardiac biomarkers: more useful in the

diagnosis of MI.
25
Cardiac enzymes
– Cardiac enzymes are markers found in the blood.

– Tested when Myocardial Infarction (MI) is suspected.

– Normally present at all times but significantly elevated

during a damage of the heart muscle.

– Common Enzymes in myocardial infarction:

Creatine kinase (CKMB)
Lactate dehydrogenase(LDH)
Aspartate Aminotransferase(AST)
 Cardiac enzymes and biomarkers:
26
– Myoglobin: Small-size heme protein not enzyme found
in all tissues mainly assists in oxygen transport often
occur more rapidly than Ti and CK.
– released from damaged tissue within 1 hour with high
sensitivity, but low specificity (can be elevated during
other muscle injuries).
– Time to rise is 1-4 hours’ Peak time 6-12 hours return to
normal in 24 hours.
– the main reason that myoglobin has not been used by
most hospitals for the evaluation of chest pain is its poor
clinical specifity (60% - 90%).
27
Troponin
– contractile protein not enzyme, two types:
– Troponin T found in the cardiac and skeletal muscle,
elevated during kidney and skeletal muscle damage early
rise after 3-4 hours, peak is 24 hours.
–Troponin I found only in cardiac muscle, more specific but
rises later,4-6 hours, peak is at 18 hours return to normal level
in 14 days so its sensitivity 100% if tested after 12 hours of
onset of chest pain but myoglobin is more sensitive in the
early hours but one of the disadvantages can’t be used to
detect re-infarction.
–Troponin can detect smaller infarction than other
biomarkers can do.
28 Creatine Phosphokinase (CK-MB):
–Creatine kinase (CK/CPK) is an enzyme expressed in a
number of tissues, it catalyses the conversion of creatine
to phosphocreatine degrading ATP to ADP.
– The CK enzyme consists of two subunits, B (brain type) or
M (muscle type), Making three different isoenzymes: CK-
MM, CK-BB and CK-MB.
29
–with CK-MB elevations as an important marker following
myocardial infarctions, elevations in CK-MM an indicator
of muscle disease, and increases in CK-BB an occasional
finding following brain infarcts.

–High specificity for cardiac tissue Begins to rise 4-6 hours

after onset of infarction Peaks at about 12 hours Returns
to baseline at 24-48 hours

– Can be used to indicate early re- infarction if level

normalizes and then increases again Normal serum value
for CK is 15- 100 U/L for males and 10-80 U/L for females
30
Lactate dehydrogenase(LDH):
–Lactate dehydrogenase catalyses the conversion of
pyruvate to lactate an essential step in the metabolic
process that ultimately produces cellular energy

–Normal level : 55-140 IU/L at 30°C

31
– The highest concentration of enzyme is located in the heart,
skeletal muscle, liver, kidney, brain, and erythrocytes. There are 5
isoenzymes of LDH.

– there is LDH1 and LDH2, normally LDH2 is greater than LDH1, if LDH1
is greater than LDH2 than the person is positive for MI.
– elevates in 24-48 hours and peaks in 48-72 hours after the episode.

– LDH levels are also high in Tissue breakdown • Hemolysis • cancer,

• meningitis, • encephalitis, or HIV so has only limited diagnostic
value because of its nonspecific nature.
– Because many common diseases increase total LDH (LD) levels,
isoenzyme electrophoresis is usually necessary for diagnosis.
32
 AST (Aspartate Aminotransferase):
33
–elevation 8-12 hours after infarction Peak levels 24-48
hours after the MI.
–This enzyme is not particularly indicative of MI.
– Other conditions can also cause a rise in the levels as in
trauma to the skeletal muscles, liver disease.
34
 Enzymes of gastrointestinal
diseases
• Amylase
35
• lipase
 Amylase
36
– catalyses the hydrolysis of starch into maltose (disaccharides).
Amylase
Starch + H2O maltose (disaccharides)

– Amylase was the first enzyme to be discovered and isolated (1833).

– Normal level of amylase go up to 140 U/L.
– It is made in the pancreas and salivary glands in humans and some
other mammals.
37 CLINICAL SIGNIFICANCES of amylase
– Used in diagnosis and monitoring pancreatitis.
• Acute: transient increase in activity with 2- 12 hours.
• Serum amylase may be raised in bile duct obstruction
And peptic ulcer disease.
• Raising amylase activity in salivary gland diseases.
38 Lipase
–Lipase is a hydrolytic enzyme, made primarily by
pancreatic acinar cells
–digests fats into glycerol and fatty acids
–A normal lipase level can range from 0-160 U/L
depending on the lab.
Lipase
Fat + H 2O Glycerol + Fatty Acids
39 –amylase or lipase levels at least 3 times above the
reference range are generally considered diagnostic of
acute pancreatitis.
– Pancreatic lipase monitoring is also used to help
diagnose: Crohn's disease, Cystic fibrosis, and Celiac
disease.
40 aldolase isozymes
–Three aldolase isozymes (A, B, and C), encoded by three
different genes, are differentially expressed during
development.
–aldolase A, which predominates in muscle; aldolase B in
liver; and aldolase C in brain.
– Aldolase A is found in the developing embryo and is
produced in even greater amounts in adult muscle.
Aldolase A expression is repressed in adult liver, kidney
and intestine and similar to aldolase C levels in brain and
other nervous tissue
 Aldolase A enzyme
41
– found bound to the actin-containing filament of cytoskeleton.
regulates cell contraction through its reversible binding to these
filaments.

– also known as fructose-bisphosphate aldolase

– catalyzes the reversible conversion of fructose-1,6-bisphosphate

to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone
phosphate (DHAP) via the glycolysis metabolic pathway .

– Aldolase A deficiency has been associated with myopathy and

hemolytic anemia.
42
Enzymes as Tumor Markers
–The most commonly used tumor markers with enzymatic
activity are:
–Prostatic acid phosphatase (PAP)
–Prostate-specific antigen (PSA)
–Alkaline phosphatase
–Neuron-specific enolase
43
Prostatic acid phosphatase (PAP),
– also prostatic specific acid phosphatase (PSAP), is an
enzyme produced by the prostate.

–The highest levels of acid phosphatase are found in

metastasized prostate cancer. Diseases of the bone,
such as Paget's disease, osteoporosis or
hyperparathyroidism, diseases of blood cells, such as
sickle-cell disease or multiple myeloma or lysosomal
storage diseases, such as Gaucher's disease, will show
moderately increased levels.
44 –Certain medications can cause temporary increases or
decreases in acid phosphatase levels. Manipulation of
the prostate gland through massage, biopsy or rectal
exam before a test may increase the level.

–Its physiological function may be associated with the

liquefaction process of semen

–PSAP was used to monitor and assess progression of

prostate cancer until the introduction of prostate specific
antigen (PSA), which has now largely displaced it
45 Prostate Specific Antigen (PSA)
–The clinical use of PAP has been replaced by PSA. PSA is
much more specific for screening or for detection early
cancer. It is found in mainly prostatic tissue.
–PSA exists in two major forms in blood circulation. The
majority of PSA is complexed with some proteins. A minor
component of PSA is free.
–The use of PSA should be together with digital rectal
examination and followed by transrectal
ultrasonography for an accurate diagnosis of cancer.
–Serum level of PSA was found to be correlated with
clinical stage, grade and metastasis
46
–The greatest clinical use of PSA is in the monitoring
of treatment.
–The PSA level should fall below the detection limit
after treatment.
–This may require 2-3 weeks. If it is still at a high level
after 2-3 weeks, it must me assumed that residual
tumor is present.
47
Medical applications of enzymes
–Development of medical applications for enzymes have
been at least as extensive as those for industrial
applications
–Streptokinase: It is a bacterial enzyme. It has fibrinolytic
action and is used to dissolve blood clots in the
treatment of myocardial infarction (MI).

–Urokinase: It is prepared from human urine. It is also used

to dissolve blood clots in the treatment of myocardial
infarction (MI) and pulmonary embolism.
48 –Pepsin: It is isolated from hog stomach and is used in
treatment of gastric indigestion.

–Fungal diastase, amylase and lipase: These enzymes are

constituents of digestive syrups and tablets and are used
in treatment of gastric indigestion and pancreatic
insufficiency (EPI).

–Asparaginase: This enzyme has been used as an anti-

tumour agent to treat adult leukaemia.

–Ribonuclease: antiviral therapy, cause RNA hydrolysis.

49
Enzyme inhibitors
– blocking an enzyme's activity can kill a pathogen or correct a
metabolic imbalance, many drugs are enzyme inhibitors. They are
also used in pesticides.

– Uses of enzyme inhibitors:

– Chemotherapy: Methotrexate blocks the action of dihydrofolate
reductase and thereby halts the production of nucleotides. This
block of nucleotide biosynthesis is more toxic to rapidly growing cells
than non-dividing cells.
– Antibiotics: Drugs also are used to inhibit enzymes needed for the
survival of pathogens. For example, Many antibiotics such as
penicillin and vancomycin inhibit the enzymes that produce the cell
membrane
50 –Metabolic control: Many metabolic pathways in the cell
are inhibited by metabolites that control enzyme activity
through allosteric regulation or substrate inhibition, When
ATP levels rise, ATP binds an allosteric site in PFK1 to
decrease the rate of the enzyme reaction; glycolysis is
inhibited and ATP production falls.

–Pesticides: Acetylcholinesterase (AChE) inhibitors are used

in both medicine (in the treatment of myasthenia gravis
and in anesthesia) and in agriculture(The
organophosphate pesticides).
51 –Natural poisons: An example of a toxic peptide is alpha-
amanitin, which is found in relatives of the death cap
mushroom. This is a potent enzyme inhibitor, preventing
the RNA polymerase II enzyme from transcribing DNA.
52 enzymopathy

–An enzymopathy may be caused by the absence of an

enzyme , a decrease in enzymes activity , or the absence
or incorrect synthesis of a coenzyme .

–Each of these abnormalities leads to specific metabolic

disorder and determines the clinical picture of the given
enzymopathy . For eg. , abnormal metabolism of
carbohydrates may be manifested by diabetes mellitus or
galactosemia, abnormal metabolism of fats, by tay sachs
disease or Niemann – pick disease, and abnormal
metabolism of amino acids , by alkaptonuria or albinism
53
Home work

–Metabolic defect in amino acid metabolism including:

–Phenylketonuria
–Maple syrup urine disease
–Albinism
–Homocystinuria
–alkaptonuria
54 enzyme deficiency due to genetic disorders
–Genetic mutation loss of enzyme activity
–Increased amount of substrate in serum
–Prenatal diagnosis in amniotic fluid
–Enzyme therapy for EDD like:
–Galactosemia
–Glu.6.phosphate dehydrogenase
–Cholenesterases
–Phenylketonuria
–Albinism, alkaptonuria
55
56 Galactosemia
–Galactosemia is a rare disorder that affect the body ability
to breakdown galactose(found in milk and milk products)
to glucose
–Galactose builds up and can cause damage to the brain,
eyes, liver and kidneys.
–autosomal recessive disorder caused by deficient or
absent activities of one of the three enzymes involved in
the galactose metabolic pathway: galactokinase,
galactose-1- phosphate uridyltransferase and UDP-
galactose 4’-epimerase.
57 Galactosemia
– Galactosemia is
treated with a
modified diet(called
the galactose
restricted diet).
58 Glucose.6.phosphate dehydrogenase

– FUNCTION OF G6PD:
– Regenerates NADPH,
allowing regeneration of
glutathione
– Protects against oxidative
stress
–Lack of G6PD leads to
hemolysis during oxidative
stress- infection,
medication, fava beans.
 Glucose.6.phosphate dehydrogenase deficiency
59
–X-linked recessive disorder that results in defective glucose-
6-phosphate dehydrogenase enzyme predisposes to non-
immune hemolytic anemia

– Most of the time, those who are affected have no

symptoms.
–Following a specific trigger, symptoms such as yellowish skin,
dark urine, shortness of breath, and feeling tired may
develop.

–Complications can include anemia and newborn jaundice.

– Some people never have symptoms.
60

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