Clinical Enzymology
Objectives
•
List of clinically important enzymes and iso-enzymes.
•
Enzymes and iso-enzymes & the tissues in which they are found
•
Diagnostic enzymes, Therapeutic enzymes and use of enzymes in clinical laboratory
to measure concentrations of other analytes
•
Enzyme changes in Myocardial infarction, Liver disease, Bone disease, Muscle
disease, Pancreatitis, Cancers
•
Biomarkers of MI
Enzymes
 Biological catalysts
 Very efficient –can increase reaction rates at the order of x 10
 Most are proteins (some RNA)- so liable to denaturation
 Specific to substrates
 Partly specific to tissues
 Assay by measure of rate of specific reaction catalyzed by that enzyme
Diagnostic enzymology
 Enzymes may be Extracellular or Intracellular (and present in low concentration in
blood)
 Serum levels of enzymes may increase due to more production/ more diffusion from
cell membrane/ cell destruction/ decreased excretion in bile/ urine
 Some enzymes are relatively organ specific: used to making1
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
 Definitive diagnosis/ Differential diagnosis/ early diagnosis/ Prognosis
 Serum enzyme measurement is fairly non invasive & it is possible to do repeated
tests
Information from enzymes measurements in serum
 Presence of disease
 Organs involved
 Aetiology /nature of disease: differential diagnosis
 Extent of disease-more damaged cells-more leaked enzymes in blood
 Time course of disease
Enzymes routinely measured
NAME OF THE ENZYME
PRESENT IN
Aspartate Amino transferase (AST)
Serum glutamate-oxaloacetate
transaminase (SGOT)
Heart and liver
Alanine Amino transferase (ALT)
Serum glutamate-pyruvate transaminase
(SGPT)
Liver
Alkaline Phosphatase (ALP)
Bone, intestine, placenta, liver
Acid Phosphatase (ACP)
Prostate
 glutamyl Transferase ( GT)
Biliary tract
Creatine kinase (CK)
Skeletal Muscle, cardiac muscle,
brain
Lactate Dehydrogenase (LDH)
Heart, liver, muscle, RBC
 Amylase
Pancreas
Isoenzymes
•
Catalyze same reactions, but are formed from structurally different polypeptides.
•
They perform the same catalytic function.
2
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
•
Different iso-enzymes may arise from different tissues and their specific
detection may give clues to the site of pathology.
•
Various iso-enzymes of an enzyme can differ in:
- physical properties (eg heat stability)
- biochemical properties such as amino acid composition and immunological reactivity.
Measurement of enzyme activity
•
Enzyme activity is expressed in International unit (IU)
It corresponds to the amount of enzymes that catalyzes the conversion of one
micromole (mol) of substrate to product per minute
LACTATE DEHYDROGENASE (LDH)

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

It exists as 5 different isoenzymes with various combinations of H and M subunits
3
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
Isoenzyme Composition Composition
name
Present in
Elevated in
LDH1
( H4)
HHHH
Myocardium,
RBC
myocardial
infarction
LDH2
(H3M1)
HHHM
Myocardium,
RBC
LDH3
(H2M2)
HHMM
Kidney,
Skeletal
muscle
LDH4
(H1M3)
HMMM
Kidney,
Skeletal
muscle
LDH5
(M4)
MMMM
Skeletal
muscle, Liver
Skeletal muscle
and liver
diseases

CREATINE KINASE (CK)
Creatine + ATP
phosphocreatine + ADP
(Phosphocreatine – serves as energy reserve during muscle
contraction)
 Creatine kinase is a dimer made of 2 monomers
occurs in the tissues
 Skeletal muscle contains M subunit, Brain
contains B subunits
 Three different isoenzymes are formed
4
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
Isoenzyme
Composition Present in
name
Elevated in
CK-1
CNS diseases
BB
Brain
CK-2
MB
Acute
Myocardium
myocardial
/ Heart
infarction
CK-3
MM
Skeletal
muscle,
Myocardium
ALANINE TRANSAMINASE (ALT) AND ASPARTATE TRANSAMINASE( AST)
 Alanine transaminase (ALT) and Aspartate transaminase (AST) enzymes are the
most abundantly present in the liver and is elevated in blood as a result of leakage
from damaged cells
 Measurement of these transaminases is useful for the diagnosis of liver diseases
 In viral hepatitis the enzyme levels are increased 20-50 times above the upper limit
of the normal range
 Alanine transaminase (ALT) increase is specific for liver damage involving
hepatocellular damage
 Aspartate transaminase (AST) is moderately increased in Muscular dystrophy and
acute myocardial infarction
5
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
LEVELS OF ENZYMES IN DISEASES
INVOLVING LIVER DAMAGE
In viral hepatitis
Rapid
rise
in
transaminases (AST &
ALT) in serum occurs
even before bilirubin
rise is seen
LEVELS OF ENZYMES IN MYOCARDIAL
INFARCTION
AST and CK rise in 6
hours following acute
myocardial infarction
HBDH
HBDH and LDH are
elevated much later and
remains high for a
longer period of days
LDH
CK
CK-MB
AST
6
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
ALKALINE PHOSPHATASE (ALP)
 Is a group of enzymes that have maximal activity at a high pH 9.0-10.5
 Widely distributed throughout the body
 High levels are seen is liver, bone, placenta and intestine and useful to assess
hepatobiliary and bone diseases
 In hepatobiliary obstruction,hepatocytes lining the biliary ducts induces the ALP
synthesis.
 High levels of ALP is indicative of extrahepatic obstruction rather than
intrahepatic obstruction
 In bones, the enzyme is derived from osteoblasts. Hence increased in bone
diseases like rickets, osteomalacia, neoplastic diseases with bone metastates and
healing fractures
ALKALINE PHOSPHATASE (ALP) conti
p-NPP + H2O
Para nitro
phenylphosphate
ALP, Mg2+
pH 10.3
p-NP (benzenoid form) + PO43Colorless
Rearrangement
p-NP (quinonoid form) + PO43Yellow
Color read at 405nm
 The
activity of the bone isoenzyme can be estimated by heat
treating a serum sample at 56oC. The bone ALP is heat
liable and is destroyed or heat
inactivated at this
temperature.
 Measurement
of ALP before and after heat treatment gives a
measure of bone ALP
7
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
ACID PHOSPHATASE (ACP)
 Is a group of enzymes that have maximal activity at pH 5.0-6.0
 It is present in prostate gland, liver, spleen and RBC.
 The main source of ACP is prostate gland and so can be used as a marker for
prostate disease.
AMYLASE
 Is the digestive enzymes from the pancreas and salivary
glands to digest complex carbohydrates.
 Elevated in acute pancreatitis.
 It is used as a marker to detect acute pancreatitis AND appendicitis.
 glutamyltransferase ( GT)
It is involved in aminoacid transport across the membranes.
Found mainly in biliary ducts of the liver, kidney and pancreas.
Enzyme activity is induced by a number of drugs and in particular alcohol.
-GT increased in liver diseases especially in obstructive jaundice.
-GT levels are used as a marker of alcohol induced liver disease and in liver cirrhosis.
MEASUREMENT OF ENZYMES
Enzymes are measured
End point assay
Kinetic assay
8
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
Measurement of enzymes are affected by the presence of inhibitors or activators.
Hence most of the enzymes are measured by coupled assay.
A coupled assay is one in which a second enzyme is used to act on the product of the
enzyme of primary interest. Second enzyme used NADH as coenzyme. The rate can be
followed by measuring oxidation of NADH which can be done conveniently at 340nm.
Principle involved in AST estimation
- Oxoglutarate + L-aspartate
Aspartate
aminotransferase
AST
L- glutamate + oxaloacetate
+
NADH + H+
Malate dehydrogenase
MDH
L-matate + NAD+
9
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
MEASUREMENT OF ENZYMES
NAME OF THE ENZYME
Conditions in which level of activity in
serum is elevated
Aspartate Amino transferase (AST)
Serum glutamate-oxaloacetate
transaminase (SGOT)
Myocardial infarction, Liver disease especially
with liver cell damage
Alanine Amino transferase (ALT)
Serum glutamate-pyruvate
transaminase (SGPT)
Liver disease especially with liver cell damage
Alkaline Phosphatase (ALP)
Liver disease- biliary obstruction
Osteoblastic bone disease-rickets
Acid Phosphatase (ACP)
Prostatic carcinoma
 glutamyl Transferase ( GT)
Liver disorder like liver cirrhosis
Creatine kinase (CK)
Myocardial infarction and skeletal muscle
disease(muscular dystrophy
Lactate Dehydrogenase (LDH)
Myocardial infarction, other diseases like liver
disease.some blood diseases
 Amylase
Acute pancreatitis
SUMMARY
 Enzymes are biological catalysts present in every cell of the body.
 An enzyme will act on a specific substrate yielding a product.
10
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
 An isoenzyme is a genetic variant produced largely within a specific tissue.
 Isoenzyme patterns can give information about organ-specific disease.
 Important enzymes in the investigation of heart disease are CK, LDH and AST.
 Important enzymes in the investigation of liver disease are AST, ALT, alkaline
phosphatase and GGT.
 Creatine kinase has three isoenzymes: CK-MM, CK-MB and CK-BB.
 LDH has five isoenzymes.
 Alkaline phosphatase can be used in the investigation of liver and bone disease.
 Increased levels of acid phosphatase are found in prostate cancer.
 GGT is induced by alcohol and is useful in monitoring alcohol abuse.
 Enzyme measurements should be performed using zero order kinetics, i.e. using
excess substrate.
 Determinations of enzyme activity can be performed using an end-point or kinetic
method
ACID PHOSPHATASE
•
Hydrolyzes Phosphoric acid ester at pH 4-6
•
Secreted by prostate, RBC, WBC, Platelets
•
Prostatic iso enzyme inhibited by Tartrate
•
Normal range 2.5-12 IU/L
•
Prostatic isoenzyme: 1 IU/L
•
Tumour marker: increased in Ca Prostate sply. with bone metastasis
•
False high reading if sample taken after rectal exam/ if sample is hemolyzed
11
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
Amylase
•
Splits Starch & Dextrin to maltose
•
Secreted by pancreas & salivary glands
•
Normal seum levels: 20-115 IU/L
•
In acute pancreatitis, serum Amylase begins to rise 2- 12 hours after onset of an
attack, peak at 24 hours, and return to normal levels within 3-5 days.
•
Moderately high levels in Chronic pancreatitis, mumps and pancreatic duct
obstruction
Lipase
•
Splits Tg to mono acyl glycerol + 2 FA
•
Pancreatic enzyme
•
More specific for Acute Pancreatitis than Amylase
•
Normal value: 50-175 IU/L
•
Lipase elevations persist for approximately 7-14 days in acute pancreatitis
•
L2 isoenzyme is most clinically specific and sensitive.
•
Moderate increase in levels in Ca Pancreas, biliary disease and perforated
peptic ulcer.
•
Normal value in Mumps
Prostate specific antigen (PSA)
•
Serine protease secreted mainly by secretory glandular epithelium of Prostate
•
Mainly secreted into seminal fluid and causes liquefaction of semen
•
Serum levels: 1-4 Micrograms/ L
•
4-10 Micrograms/ L : BPH
 10 Micrograms/ L : Ca Prostate
12
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
Brain natriuretic peptide
•
1 of 3 natriuretic peptides responsible for preventing excess salt & water
retention:
1. Atrial natriuretic peptide (ANP): secreted by cardiac atria
2. Brain natriuretic peptide (BNP): secreted by cardiac ventricles & brain
3. C-type natriuretic peptide (CNP): unknown function
•
BNP secreted in response to excessive stretching of ventricular walls.
•
In Congestive cardiac failure ANP & BNP are high. BNP correlates with
ventricular dysfunction & high values indicate poor prognosis
NUERON SPECIFIC ENOLASE (NSE)
•
Iso-enzyme of Enolase found in neural tissue & APUDOMAS
•
Glycolytic enzyme
•
Tumour marker: raised in cancers of Neuro-endocrine origin:
1. Small cell cancer of lungs,
2. Neuroblastoma
3. Pheochromocytoma
4. Medullary cancer of Thyroid
13
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
14
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
CARDIAC MARKERS
•
CK
•
CK-MB
•
CardiacTroponin I (cTn I)
•
CardiacTroponin T (cTn T)
•
Myoglobin
•
AST / SGOT
•
LDH
•
LDH 1
15
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
•
16
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
MYOGLOBIN
•
Oxygen carrying heme-protein of muscles
•
Leaks into blood after injury to cardiac or skeletal muscle and is seen in urine
because of its small molecular weight
•
Early indicator of MI
•
Highly sensitive, but non-specific marker
•
Appears in urine 1-3 hrs after MI, peaks after 6-9hrs and disappears after24
hrs.
•
Non-enzymatic marker
Cardiac Troponins
•
There are 3 Troponins in a Troponin complex:
1. Troponin I: Actomyosin ATPase inhibitory subunit
17
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
2. Troponin T: Tropomyosin binding subunit
3. Troponin C: Calcium binding subunit
•
Troponin I has 3 isoforms: slow-skeletal, fast-skeletal and cardiac.
•
Cardiac isoforms of Tn I & Tn T are cTn I & c Tn T respectively & they are
cardio specific
•
Normal value cTn I & c Tn T: 1-10 Micrograms/L
•
Rise above upper limit 3-8 hrs after MI,
•
c Tn I peaks after 24-48 hrs, returns to normal after 3-5 days
•
c Tn T peaks after 72-100 hrs, returns to normal after 5-10 days
•
Non-enzymatic early markers
•
Sensitive & Specific
Creatine Kinase (CPK)
•
Normal range: 15 – 160 U/L (CK-MB <6% total)
•
Raised in Muscle injury/ disease, MI, Injury/ tumour of Brain
•
Exceeds upper limit of normal range 3-8 hrs after MI, peaks after 10-24 hrs,
returns to normal after 3-4 days
CK – MB
•
Normal range: 0-6 IU/L
•
Raised in MI, myocardial injury/ischemia, angina, cardiac surgery, Duchennetype muscular dystrophy, polymyositis,malignant hyperthermia, Reye’s
syndrome, CO poisoning
•
Exceeds upper limit of normal range 3-8 hrs after MI, peaks after 10-24 hrs,
returns to normal after 3-4 days
18
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
AST
•
Normal Range: 5 – 34 IU/L
•
Rises above upper limit of normal value 24-36 hrs after MI, peaks after 4-5
days, returns to normal after 10-12 days
•
Late marker, non specific, no longer used
Lactate Dehydrogenase
•
N. Value: 150 - 300 IU/L
•
Normally LDH 2> LDH 1
•
After MI, LDH1/LDH2 > 1 (FLIPPED PATTERN)
•
LDH and LDH 1 rise above upper limit of normal 8-12 hrs after MI, peak
after 72-144 hrs, return to normal after 8-14 days
•
Late marker, non specific, no longer used
DIAGNOSIS OF MI BASED ON CK & LDH PATTERNS
•
CK MB > 6% Total CK; LD 1 > LD 2
Reliable diagnostic criteria for AMI
•
CK MB > 6% Total CK; LD 2 > LD 1
Myocardial damage with/ without AMI
•
CK MB < 6% Total
No MI, regardless of LD
19
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
20
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.
Summary of Cardiac Markers
MARKER
RISES ABOVE UPPER
LIMIT OF NORMAL
RANGE
PEAK
RETURNS TO
NORMAL
CK
3-8 HRS
10- 24 HRS
3-4 DAYS
CK MB
3-8 HRS
10- 24 HRS
2-3 DAYS
LDH, LDH 1
8-12 HRS
72- 144 HRS
8-14 DAYS
MYOGLOBIN
1-3 HRS
6- 9 HRS
24 HRS ( 1 DAY)
C TnT
3-8 HRS
24- 48 HRS
3-5 DAYS
CTnI
3-8 HRS
72- 100 HRS
5-10 DAYS
21
’ Clinical Enzymology & Cardiac Markers’ Handout for 1st yr MBBS by Dr. Renu Nagar, Dept of
Biochemistry, Dr. RPGMC, Tanda.