Clinical Enzymology
Measurement of serum
enzymes
Enzymes are normally intracellular and LOW
concentration in blood
Enzyme release (leakage)in the blood indicates cell
damage (cell death, hypoxia, intracellular toxicity)
Quantitative measure of cell/tissue damage
Fairly non invasive possible to do repeated tests
Organ specificity- but not absolute
specificity in spite of same gene content.
Most enzymes are present in most cellsdiffering amounts
Information from enzymes
measurements in serum
Presence
Organs
of disease
involved
Aetiology
/nature of disease:
differential diagnosis
Extent
of disease-more damaged
cells-more leaked enzymes in blood
Time
course of disease
Enzymes in Liver disorders
AST
ALT
ALP
LDH
glutamyltransferase ( GGT)
glutamate dehydrogenase (GLD)
5'-nucleotidase
Cholinesterase (CHE)
Liver Tests
AST, ALT
Alkaline Phosphatase
GGT
Bilirubin
True liver function tests
Albumin
PT/INR
Deferential diagnosis of liver
disorder
Hepatocellular (functional disorder)
Obstructive liver disorders
(mechenical)
Hepatocellular predominant
AST & ALT increased
Enzymes that are in the hepatocyte and
function during gluconeogenesis
Leak out of the hepatocytes in times of
injury and can be measured in the serum
Both enzymes require P5P (vitb6) as
coenzyme
The deficiency of P5P affect ALT more than
AST (what seen in alcoholic)
Normally present in serum at levels ~30-40
U/L
AST/ALT
AST:
liver > cardiac muscle > skeletal muscle >
kidney > brain > pancreas > lung > leukocytes
> erythrocytes
Less specific for liver damage
Can increase with strenuous exercise, MI
Located in cytosol and mitochondria of
hepatocytes
Cleared more rapidly than ALT
ALT
Mainly from cytosol of hepatocytes
more specific for liver damage
LEVELS OF ENZYMES IN DISEASES INVOLVING
LIVER DAMAGE
In
viral
hepatitis
Rapid rise in
transaminase
s (AST & ALT)
in
serum
occurs even
before
bilirubin rise
is seen
Aspartate Aminotransferase
(AST , GOT)
Diagnostic Significance:
Hepatocellular disorders:
Viral hepatitis (100 times ULN in
acute hepatitis)
Cellular damage
Liver cirrhosis (Moderate increase ,
4-8 ULN)
Chronic viral hepatitis
Chronic alcoholism
Necrosis
Alanine Aminotransferase
(ALT , GPT)
Diagnostic Significance:
Hepatocellular disorders:
Viral hepatitis
Cellular damage
Necrosis
Serum aminotransferace levels in
various liver disease
LACTATE DEHYDROGENASE
(LDH)
Pyruvate
glycolysis)
Lactate
(anaerobic
LDH is elevated in myocardial infarction,
blood disorders
It is a tetrameric protein and
made of
two types of subunits namely H =
Heart, M = skeletal muscle (4 peptide
chains of 2 types)
It exists as 5 different isoenzymes with
various combinations of H and M subunits
Isoenzym Compositi
e name
on
Compositio Present in
n
LDH1
HHHH
( H4 )
Elevated in
Myocardiu myocardial
m, RBCHaemolysis
infarction
(megaloblastic,
pernicious anemia)
LDH2
(H3M1)
HHHM
Myocardiu
m, RBC
LDH3
(H2M2)
HHMM
Kidney,
Skeletal
muscle
LDH4
(H1M3)
HMMM
Kidney,
Skeletal
muscle
LDH5
(M4)
MMMM
Skeletal
muscle,
Liver
Skeletal
muscle and
liver
diseases
Cholestatic
ALKALINE
predominance
PHOSPHATASE (ALP)
Alkaline phosphatase (ALP) belongs to
a group of enzymes that catalyze the
hydrolysis of various
phosphomonoesters at an alkaline pH
(9~10)
Liberate inrganic phosphate from
organic phosphate
Activators: Mg . Mn
Alkaline Phosphatase
Exists in liver in membrane of
hepatocyte where it lines the
canaliculus
ALKALINE PHOSPHATASE (ALP)
Tissue sources:
Liver
Bone
Intestine
Placenta
Spleen
Kidney
ALKALINE
PHOSPHATASE (ALP)
Diagnostic significance
Hepatobiliary disorders
Obstructive jundice ( obstruction
induces synthesis of ALP by
hepatocytes)
The elevation is always greater in
extrahepatic obstruction than
intrahepatic obstruction.
Other cholestatic enzymes
GGT: gammaglutamyltransferase
Found in hepatocytes and biliary
epithelial cells
5 nucleotidase
Both these enzymes can be used to
confirm alk phos elevation is coming
from liver
GGT is also sensitive to alcohol
ingestion
Cardiac muscular
disorder enzymes
CK
LDH
AST
Enzymes used in the diagnosis
of AMI
CK
Diagnostic significant:
Cardiac disorders
Muscle disorders (Muscle
dystrophy)
CNS disorders
Cerebrovascular accident
Seizure
CK isoenzymes in Normal sera
CK MM is major (94 ~ 100%)
CK MB less than 6%
Ck BB very small quantity
CK isoenzymes measurements
Electrophoresis
Ion exchange chromatography
Immunoassay
Immunoinhibition
CK
Macro CK : Ck BB complexed with IgG
or IgA
CK Mi : Mitochondrial CK
CK MB measurement
Immunoinhibition method?
anti CK-M subunit is added to reagent
This inhibits CKMM and M subunit of
the CKMB
This activity of CKBB and B sub uint of
CKMB only measured
CK BB activity is absence in the serum
Multiply the B sub unit of CKMB by 2
to determine the activity of CKMB
Not specific (Macro CK inteference)
Pancreatic enzymes
PANCREATIC ENZYMES
serum AMY
lipase (LPS)
Trypsin (TRY)
Chymotrypsin (CHY)
elastase 1
a-Amylase
Hydrolase class
hydrolysis of l,4-a-glucosidic linkages in
polysaccharides (breakdown of
glycogen and starch into glucose ,
maltose and dextrin)
Small molecule enzyme (normally can
found in urine)
Found mainly in salivary gland (S type)
and pancrease (P type)
AMS in Acute pancreatitis
Elevate within 5 to 8 hrs of onset and return
to normal in 3 to 4 days
Increase in both serum and urine
Non specific finding
Elevates in other condintion
Clinical specificity : 20% to 60%
To increase specificity:
Urine AMS
AMS clearance study
AMS isoenzymes
LPS measurment
AMS isoenzymes
P isoamylase (p1, P2 , P3) : from
pancreases
S isoamylase (S1, S2, S3) : from salivary,
fallopian tube and lung
S isoamylaese migrates more faster in
electrophoresis
In acute pancreatitis : P3 is most abundant
Salivary AMS can be inhibited by wheat
germ lectin
Lipase
Lipases are defined as enzymes that
hydrolyze glycerol esters of long-chain fatty
acids
LPS concentration in the pancreas is about
5000-fold greater than in other tissues
Most of the LPS activity found in serum
derives from the pancreas.
totally reabsorbed by the renal tubules,
and it is not normally detected in urine
The e complete absence of LPS has been
reported. Such congenital absence results
in fat malabsorption and severe
to diagnose acute pancreatitis
More specific than AMS ( elevation
persist for more than 5 days).
increases within 4 to 8 hours,
peaks at about 24 hours, and
decreases within 8 to 14 days
Obstruction of the pancreatic duct by
a calculus or by carcinoma
TRYPSIN
The acinar cells of the human pancreas
synthesize two different trypsins (1 and 2) in
the form of the inactive proenzymes(or
zymngens)
trypsinogens are converted to active TRY, a
smallpeptide is cleaved from the N-terminal
region of trypsinogen(trypsinogen activation
peptide or TAP)
TAP ,easurment may provide useful
information onthe severity of acute
pancreatitis
Clinical significant of TRY
Trypsin-1 :
In acute pancreatitis, serum TRY-1
rises in parallel with serum AMY
activity
Renal failure
cystic fibrosi (neonates)
Trypsin-2:
In acute pancreatitis (more than TRY1)
CHYMOTRYPSIN
serine proteinase. It hydrolyzes peptide
bonds involving carboxyl groups of Trp,
Leu, Tyr, or Phe, with preference for the
aromatic residue
The acinar cells of the human pancreas
synthesize two different chymotrypsins
The CHY activity in stool is in used for
the investigation of chronic pancreatic
insufficiency.
ELASTASE-1
serine proteases
It is a carboxyendopeptidase that
catalyzes hydrolysis of native
elastin, the major structural fibrous
protein in connective tissue
El measurement in stool is the
most reliable and sensitive
noninvasive procedure for the
diagnosis of chronic pancreatic
insufficiency
Bone
Enzymes
Bone ALP
(tartrate-resistant acid
phosphatase)
ACID PHOSPHATASE (ACP)
Acid phosphatase (ALP) belongs to a
group of enzymes that catalyze the
hydrolysis of various
phosphomonoesters at an acid pH (5 )
Belong to hydrolase class of enzymes
(same as ALP)
ACID PHOSPHATASE (ACP)
Tissue source:
Prostate , bone , liver, spleen ,
erytrocytes, platlets.
Diagnostic significant:
Prostatic cancer, prostatic
hyperplasia , prostatic surgery
Bone disorders ( TR-ACP activity):
Pagets disease
Hyperparathyrodism
Bone malignance
Osteomalacia
The only non bone condition in which
elevated activitiesof TR-ACP are found in
serum is Gaucher's disease of spleen, a
lysosomal storage disorder
ACID PHOSPHATASE
(ACP)
Methods of enzyme assay:
Total ACP are measured using same
methods of ALP BUT performed in
acid pH
ACID PHOSPHATASE (ACP)
Measurement of prostatic ACP
Chemical inhibition using tartarate. 1
The prostatic fraction is inhibited
by tartarate
Total ACP - ACP after tartrate inhibition
= prostatic ACP
2. using substrate specific for
prostatic ACP
thymolphthalein monophosphate
