Diagnostic Enzymology
Fourth Year Medicine
 Enzymes can be used as “markers” to detect and localize
cell damage or proliferation [(1) enzymes are present at
much higher concentrations inside cells than in plasma,
(2) some enzyme occur predominantly in certain types of
cell]
 In “normal” healthy individuals (where there is a steady
cell
turnover),
enzyme
activity
in
plasma/serum
represents: a balance between its rate of liberation into
the
Extracellular
(E.C.)
space
and
its
rate
of
clearance/uptake from E.C. space (+ auto degradation of
enzyme)
 General cases in which [Enzymes] in plasma are
elevated:
a. Cell proliferation: There is an increase in cell
turnover.
b. Non-proliferative increase in the rate of cell
turnover.
c. Cell damage.
d. Enzyme induction.
 Mechanisms of Elevated Enzyme Activities:
Cell membrane crucial function in retaining cytosolic
enzyme within cells.
1. ATP role (maintenance of cell permeability):
In the absence of added ATP to medium containing
cells, the rate of escape of intracellular enzyme is
strictly related to the rate of depletion of
intracellular ATP.
Anoxia (e.g. in severe cardiac / respiratory disease)
may allow escape of intracellular enzyme from cells
even in the absence of organ damage.
Hypoxia (e.g. during severe exercise) may allow
transient escape of enzymes from muscle cells.
2. Drugs / Poison:
 Inhibitors of energy-yielding processes (e.g.
chlorpromazine and promethazine), exert direct
effect on cell membrane to accelerate enzyme
release.
 Bacterial toxins (e.g. generalized septicemia)
cause increases in serum enzyme activities.
** The role of electrolytes and osmotically active
molecules in maintaining cell integrity is evident.
∴ One can see why in some cases serum
enzymes elevations are non-specific. ?!
Hence: Look for indices directly related to
actual cell necrosis:
*
Mitochondrial enzymes: Only liberated
during cell necrosis e.g. Glu-DH, AST (exist
in
mitochondrial
cytosolic form).
form,
distinct
from
3. Lipid perioxidation: affect release of membrane –
associated enzymes
4. Hepatic (microsomal) enzyme induction: ALP,
GGT, 5-nucleotidase are elevated in epileptic
patients taking anti-convulsants. Also, in biliary
obstruction: bile acids promote liberation of
enzymes from damaged hepatic cell
5. The enzyme rate of clearance and elimination
Half-lives of: ALT- 6.3 days
AST- 2.0 days
CK- 1.4 days
The rate of enzyme clearance depends on its rate of
elimination from serum. Different mechanisms apply:
 Amylase and lipase
Urine (detection of urinary
amylase is essential in acute pancreatitis)
 ALP excreted partly in the bile.
 Reticuloendothelial system clears some enzymes.
 Proteolytic degradation.
 Non-biologic decay (same as where serum specimens are
not kept refrigerated).
 Conjugation of enzyme with Abs (enzyme loses its
biological potency) e.g. CK/LDH
Measurement of Enzyme Activities
Enzyme units: IU: µmol of substrate transformed per
minute under standard conditions.
New unit: Katal: amount of activity that convert one
mole of substrate/second (1 Katal = 6x107 IU)
Some Important Enzymes Clinically:
Acid phosphatase (ACP): hydrolysis of a wide range
of phosphate ester bonds at acid pH.
 The activity of ACP decreases rapidly at room
temperature unless pH is reduced below 6.0.
Isoenzyems of ACP: a series of ACPs exists in all
body cells. Soluble form in the cytosol, but the
lyosomal is the predominant form.
Important tissue sources: RBCs, platelets, Prostate.
Prostate enzyme is sensitive to L-tartrate
RBC enzyme is sensitive to formaldehyde (* in
hemolyzed blood sample in the presence of
formaldehyde ACP release is inhibited).
Other tissue forms of ACP are sensitive to L-tartrate
(detectable
in
females
and
in
males
after
prostatectomy).
Clinical Applications:
The main use of ACP lies in the diagnosis of prostatic
cancer (only when the disease has spread to adjacent bone
e.g. pelvis). The enzyme in serum is markedly increased.
 Spread to soft tissue: high ACP level may be seen.
 If tumor remains as nodule / not extended beyond
prostate capsule: only ½ of patients may show elevated
serum activity of ACP.
Therefore, ACP use as
diagnostic and screening test is limited in prostatic
cancer.
 The use of “prostate specific antigen” (PSA) is a
recent method for P.C. diagnosis and screening.
 Prostatic massage: rectal examination may cause
(rarely) transient elevation of ACP.
 Because
of
enzyme
location
predominantly
in
lysosome, raised serum ACP activity is expected in
some liver storage diseases (e.g. Gaucher’s disease,
Niemann-Pick disease).
 Acute retention of urine and passage of catheter can
cause raised serum ACP activity.
 ACP may be elevated in thromboembolic and myeloproliferative disorders.
Alkaline Phosphatase (ALP):
Low substrate specificity catalyzes hydrolysis of
phosphate esters at alkaline pH.
All ALPs contain Zn and Ser at active site.
Cellular Location:
1) Brush borders of proximal convoluted tubule of
kidney and intestinal mucosa.
2) Other membranes include: Sinusoidal and Canalicular
surfaces of hepatocytes. Its location in membranes
plays a role in absorption and transport processes.
3) Bones: ALP role in minerlization since its activity
changes with osteoblastic function.
Isoenzymes:
Placental and small intestinal mucosa: ALP forms under
separate genetic control.
Bone, liver and kidney: ALP is a product of the same
gene locus but subject to post-translational modification
within each tissue.
Θ
Liver
•
Bone
•
Placenta
•
Intestine
•

Effect of Heat:
Placental ALP is unaffected at 65˚ C, whereas all other
ALP isozymes lose their activity totally. Heating at 56˚ C
for 10 minutes: Intestinal ALP loses 20% of its activity,
liver ALP 60% and bone ALP 80%. Urea denaturation
(2M): Placental ALP most stable, bone ALP the least.
Physiological causes for raised serum ALP:
1. In infancy due to predominance of bone ALP until
about the age of puberty (2-2 ½ x adult normal). In
adult most ALP activity is due to liver isoenzyme.
2. Intestinal ALP appears only in serum if a person is of
blood groups O or A, secretors of ABH red cell
antigens and are positive for Lewis antigen.
3. At puberty: up to 5-6 x adult age.
4. Pregnancy: placental ALP appears in serum of
pregnant women only during second and third
trimester. Sharp reduction in P.ALP often indicates
placental insufficiency and death of fetus.
Clinical Applications:
1. Hepatobiliary Disease
A. Very high levels of ALP are seen in biliary
obstruction
Moderate levels of ALP are seen in parenchymal
liver disease
 This is important in differential diagnosis of
hepatic and obstructive jaundice.
B. In extrahepatic obstruction: serum bilirubin and
ALP rise and fall in parallel.
C. In acute hepatic necrosis there is little change in
ALP even when bilirubin concentration is rising
dramatically. If icterus fails to clear and patients go
into cholestatic phase, ALP rises substantially.
D. In biliary cirrhosis: high level of ALP even when
bilirubin is normal. However, in portal cirrhosis
ALP level remains normal.
E. In alcoholic fatty liver: ALP is raised.
F. In carcinoma or lymphatous infilteraton of liver, the
Regan isoenzyme (resembles placental ALP) has
been detected in 1% - 3% of such patients
(especially in bronchial carcinoma).
There are
various tumor–associated ALP but their importance
is in providing insight into genetic changes
occurring during neoplasia, rather than as aids to
diagnosis.
2. Bone Disease
A. Primary bone tumors (e.g. osteogenic sarcoma) and
secondary osteoblastic bone tumors (e.g. those
originating from prostate): can cause very high ALP
level. However, osteolytic secondaries show slight
to moderate increase of ALP.
B. Paget’s disease: high ALP.
C. Osteomalacia and rickets (vitamin. D deficiency):
high ALP.
D. Primary
hyperparathyroidism
with
bone
involvement: often associated with high ALP.
E. Secondary
hyperparathyoidism,
as
in
renal
ostesodystrophy: Dramatic increase in ALP.
F. Healing fractures: high ALP.
3. Intestinal disease
A. High level of ALP (bone) found after gastrectomy
due to impaired intestinal absorption of Ca++, PO4--,
and vitamin D.
Creatine Kinase (CK):
Creatine + ATP
Creatine – P + ADP
Tissues: mainly brain, skeletal and smooth muscles.
Physiological increase:
Males > Females, increase with age and body weight
Neonates but fall rapidly during the first weeks. Physical
exercise. Labour and parturition.
Isoenzymes: 1. BB- Brain: moves rapidly towards 
2. MM- Skeletal / Heart muscles: slow
moving
3. MB- Comprises 20-40% of CK found in
heart also found in small amounts in red
fibers. Other isoenzymes e.g. x.y.z.
Clinical Applications:
1. Myocardial Infarction (M.I.): The most useful test in
establishing / refuting M.I. Increase in serum CK after 6
hours of M.I. onset reaching a peak after 36 hours,
declining to normal value after day 3.
Note:
Re-infarction,
thromboembolism,
and
arrhythmias may cause a delay in return to normal
value or cause secondary spikes.
 The following conditions can also cause moderate
increase in serum level of CK:
A. Prolonged chest pain with transient ST-segment
and T-waves changes (40% of cases).
B. Prolonged chest pain with E.C.G. abnormalities
(20% of cases).
C. Classic angina pectoris (Occasionally).
The significance of MB isoenzyme in M.I. (prognostic
Value):
There is about 2% of CK present in normal serum is of
MB-type. After infarct within few hours MB increases
dramatically but with shorter duration (due to its rapid
clearance from vascular space). The increase in the amount
of CK-MB determines the severity of M.I. and hence
prognosis.
2. Muscle Disease:
A. Duchenne Muscular Dystrophy: CK is elevated
mainly CK-MM.
Other enzymes are elevated:
Aldolase, LDH, aminotransferases.
B. Becker Muscular Dystrophy: more benign.
forms of muscular dystrophy: lesser elevation.
C. Polymyositis.
Other
D. Secondary diseases of muscles: e.g. neurogenic
atrophy and malignant hyperpyrexia.
3. Cerebral Disease:
A. Epileptic seizure due to muscular activity.
B. Tetanus
C. Organic neurologic disease e.g. cerebral infarction,
meningitis and cephalitis (Ck - BB).
4. Other conditions: elevated in alcohol intoxication,
hypothyroidism.
Lactate
Dehydrogenase
(LDH)
(NAD-
Oxidoreductase): Zn-containing enzyme, catalyzing the
following reversible reaction:
pyruvate + NADH+H+⇌Lactate + NAD+
Tissue Distribution: throughout all body tissues (in
cytosol) with high concentrations in: heart, skeletal
muscle, liver, kidney, brain, RBCs. Therefore, it is a
non-specific index of cell damage. Since its presence in
RBCs, hemolysis or delayed separation of plasma cause
artefactual increase activity of LDH.
Isoenzymes: 5: LD1
LD5
LD1 (H4), LD2 (H3M), LD3 (H2M2), LD4
(HM), LD5 (M4).
LD1: moves fastest to, while LD5 is the
slowest.
Clinical Applications:
1. Myocardial Infarction (M.I.): (>5 x normal).
 Elevation commences 12-18 hours after onset of
symptoms, peak on the third day; the activity
then declines until reaching normality by the 10th
day (in uncomplicated cases).
 LDH assay is more valuble in a case
presenting several days after suspected
M.I.
 Heart Muscle is rich in LD1 and LD2 (LD1>LD2):
In normal serum LD2> LD1
 In M.I. “flipped” pattern of LD occurs: LD1>LD2
in serum.
2. Hepatobiliary Disease:
 Increased serum LDH in acute liver necrosis.
 LDH levels are raised in most forms of
hepatobiliary
disease
including
obstructive
jaundice, and their discriminatory capacity is
poor. LD5 is the predominant isoenzyme.
3. Blood Disease:
 Marked elevation (>5x) in megaloblastic anemia,
and leukemias. Main increase in LD1 and LD2
(red nucleated cells in bone marrow is the source
of the increase).
 In hemolytic anemia LD1 and LD2 are liberated
directly from circulating RBCs.
4. Cancer:
 High serum LDH is associated with widespread
metastases especially to liver. LD4, LD5 are the
predominant isoenzymes.
The Aminotransferases (ALT/AST):
 Ala + α KG
ALT
Pyruvate + Glu
 Asp + α KG
AST
oxloacetate + Asp
Tissue Distribution:
ALT: Almost exclusively found in the cytosol and in
high concentration in hepatocyte.
AST: found in cytosol and mitochondria. Abundant in:
liver, heart, and skeletal muscle.
Clinical Applications:
1. Myocardial Infarction:
AST (markedly elevated >10x) is usually detectable
6-12 hours after onset of M.I., reaching a peak 20-48
hours after onset of symptoms, falls thereafter,
returning to reference value by the 5th day. Delayed
fall in ASP activity may be due to: heart failure.
In very severe M.I. mitochondrial AST may be
detected in serum (prognostic index).
 ALT (moderately elevated) in uncomplicated M.I.
In event of cardiac failure, severe shock or other
complications, ALT may rise >10x normal value.
This is attributed to release of ALT from damaged
hepatocytes due to tissue anoxia.
 ALT is of little diagnostic value in M.I. but
useful in detecting the complication that
develop later.
2. Hepatobiliary Disease:
 In viral hepatitis (V.H.): Both ALT and AST are
markedly increased (10-100 x normal)
 Elevation starts from prodormal phase up to 10
days before icterus (jaundice) is evident.
 ALT and AST are useful in studying the
epidemiology of V.H.
 In early genesis of V.H.: Serum AST>ALT since
[AST] in liver is higher than [ALT]. Later, serum
ALT>AST due to slower clearance of ALT (λ ½ =
6.3 days)
 Continuing elevation of serum ALT and AST is
indication of the presence of: Chronic active
hepatitis.
 If elevation is moderate, serum AST: ALT < 1:
Chronic persistent hepatitis is more likely.
 If elevation is higher than moderate and AST:
ALT > 2: Chronic aggressive hepatitis is likely
and prognosis is less favorable.
 In Portal cirrhosis: Both ALT/AST is normal if
icterus is absent, and slightly raised if present.
Elevation
rarely
more
than
3x
normal
(AST>ALT).
 In biliary cirrhosis: morderately pronounced (up
to 4 x n) increase (ALT=AST)
 In hepatobiliary obstruction: range of AST/ALT
is 2-8 x normal (depending on degree of icterus).
When obstruction is caused by non-malignant
disease, ALT>AST (usually).
In a proportion of
cases, where jaundice is caused by tumor AST>ALT.
In hepatic metastases half of patients show AST is
the dominant aminotransfrase.
Major disadvantage of Aminotransferases: Lack of
Specificity.
Other enzymes of diagnostic importance: 5 NT, GGT,
plasma and urinary -amylase, lipase, trypsinogen and
trypsin.