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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