Chapter Outline

V.

Enzymes of Clinical Significance

MI Profile
1. CK
2. AST
3. LDH
B. Liver Enzymes
1. ALT
2. ALP
3. GGT
A.

C.

Pancreatic
Enzymes
1. AMS
2. LPS
Other Enzymes
1. ACP
2. G-6-PDH

D.

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. Creatinine Kinase (CK)

Storage of high-energy creatine

phosphate in muscle cells

Highest activities in skeletal muscle,

heart (AMI), and brain tissue

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. Creatinine Kinase (CK)

Methods of Determination
i.
Forward Reaction (Tanzer-Givarg)
ii.
Reverse Reaction (Oliver-Rosalki)

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. Creatinine Kinase (CK)

Methods of Determination
i.
Forward Reaction (Tanzer-Givarg)
Measure in absorbance at 340 nm
Optimum pH is 9.0

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. Creatinine Kinase (CK)

Methods of Determination
i.
Forward Reaction (Tanzer-Givarg)
Coupled-enzyme
assay
Auxiliary
enzyme
Indicator
enzyme

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. Creatinine Kinase (CK)

Methods of Determination
ii.
Reverse Reaction (Oliver-Rosalki)
in absorbance at 340 nm is
determined
6x faster than forward reaction
Optimum pH: 6.8

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. Creatinine Kinase (CK)

Methods of Determination
ii.
Reverse Reaction (Oliver-Rosalki)

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. Creatinine Kinase (CK)

Methods of Determination
i.
Forward Reaction (Tanzer-Givarg)
ii.
Reverse Reaction (Oliver-Rosalki)

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. Creatinine Kinase (CK)

Source of Error

Hemolysis cause false CK due to AK

activity

CK is inactivated by light

Physical activity and IM injections

cause CK

Reference Range

Male, 15-160 U/L : Female, 15-130 U/L

CK-MB: <6% of total CK

Enzymes
V.

Enzymes of Clinical Significance

MI Profile

A.

Creatinine Kinase (CK)

Diagnostic Significance of CK
Isoenzymes
CK-3 / CK-MM /
CK- 2 / CK-MB /
CK-1 / CK-BB /
1.

Muscle type

Hybrid Type

Slowest mobility
2nd fastest
Major isoenzyme
Significant
in striated muscle quantities in heart
and normal serum
tissues

Brain Type

Migrate fastest
Highest
concentration in
CNS, GI tract and
uterus
(pregnancy)

Enzymes
V.

Enzymes of Clinical Significance

MI Profile

A.
1.

Creatinine Kinase (CK)

Diagnostic Significance of CK Isoenzymes

After MI, CK-MB (>6%) begin to rise

within 4-8 hrs, peak at 12-24 hrs, and
return to normal in 48-72 hrs.

V.

Enzymes of Clinical Significance

MI Profile

A.
1.

Creatinine Kinase (CK)

Diagnostic Significance of CK
Isoenzymes

Reference
Values:
94-98% CK-MM
2-6%
CK-MB

Normal Control

Myocardial
infarction

Separation of CK isoenzymes by
electrophoresis

Enzymes
V.

Enzymes of Clinical Significance

MI Profile

A.
1.

Creatinine Kinase (CK)

Other CK Isoenzymes
a.
Macro-CK
Migrate midway CK-MM and CK-MB
CK-BB complexed with IgG/IgA
CK-MM with LPP
b.
Mitocondrial CK (CK-Mi)
Migrates cathodal to CK-MM
Bound to mitochondrial membranes

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
2. Aspartate Aminotransferase (AST)

Serum glutamic-oxaloacetic transaminase

(SGOT)

Transfer of amino group in aspartate and keto acids

Involved in the synthesis and degradation of
AA.
Highest activities in cardiac, liver and skeletal
muscle.

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
2. Aspartate Aminotransferase (AST)

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
2. Aspartate Aminotransferase (AST)

Diagnostic Significance

AST levels begin to rise in 6-8 hours,

peak at 24 hours, and return to normal
in 5 days.

Also in hepatocellular and skeletal

muscle dis.

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
2. Aspartate Aminotransferase (AST)

Assay for Enzyme Activity

Karmen Method

Uses malate dehydrogenase and

monitors in absorbance at 340 nm

Falsely in hemolyzed sample

Reference Range: 5 30 U/L

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
3. Lactate Dehydrogenase (LDH)

Interconversion of lactate and pyruvate

Widely distributed, highest activities in

heart, hepatic, skeletal muscle and RBC

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
3. Lactate Dehydrogenase (LDH)

Assay of Enzyme Activity

a.
Wacker method
Forward Reaction (Lactate
Pyruvate)
b.
Wrobleuski La Due
Reverse Reaction (Pyruvate
Lactate)

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
3. Lactate Dehydrogenase (LDH)

Assay of Enzyme Activity

a.
Wacker method

Forward Reaction (Lactate Pyruvate)

in absorbance is monitored at 340 nm
Optimal pH is 8.3 8.9

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
3. Lactate Dehydrogenase (LDH)

Assay of Enzyme Activity

b.
Wrobleuski La Due

Reverse Reaction (Pyruvate Lactate)

in absorbance is monitored at 340
nm
Optimal pH is 7.1 to 7.4

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
3. Lactate Dehydrogenase (LDH)

LD begin to rise within 10-24 hrs,

peak at 48-72 hrs, and remains
elevated for 10 days.

Reference Range: 100-225 U/L

Enzymes
V.

Enzymes of Clinical Significance

MI Profile
3. Lactate Dehydrogenase Isoenzymes (LDH
Isoenzymes)

Tetramer containing two active sub-units

Lactate Dehydrogenase (LDH) Isoenzyme
Isoenzyme
Tissue
Disorder ()
A.

LDH-1
(HHHH)
LDH-2
(HHHM)
LDH-3
(HHMM)

Heart, RBC
Lung, Spleen,
Pancreas

MI, Hemolytic
anemia
RI, Megaloblastic
anemia
Pulmonary embolism

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
3. Lactate Dehydrogenase (LDH)

Relative concentration in normal

serum:

LDH-2>LDH-1>LDH-3>LDH4>LDH-5

In AMI and intravascular hemolysis,

LDH-1 and LDH-2 demonstrate a
Flipped pattern (LDH-1 > LDH-2)

Enzymes
V.

Enzymes of Clinical
Significance
A.

MI Profile
3. Lactate Dehydrogenase (LDH)

Significance of Isoenzyme
fractions

Enzymes
V.

Enzymes of Clinical Significance

A.

MI Profile
1. CK
2. AST
3. LD

Appearan
ce
Peak
Stay
Elevated

CK-MB

AST

LDH

4-8 hrs

6-8 hrs

10-24 hrs

12-24 hrs

24 hrs

48-72 hrs

3 days

5 days

10 days

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
1. Alanine Aminotransferase (ALT)

Serum glutamic-pyruvic transaminase

(SGPT)

Transfer of an amino group between

alanine and -ketoglutarate

Increased in hepatocellular disorders

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
1. Alanine Aminotransferase (ALT)

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
1. Alanine Aminotransferase (ALT)

De Ritis Ratio

The AST/ALT Ratio

Differentiates the cause of hepatic

disorder

Ratio > 1
Ratio < 1

Non viral origin

Viral in origin

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
1. Alanine Aminotransferase (ALT)

Assay for Enzyme Activity

Uses Lactate Dehydrogenase and

monitors decrease in absorbance at
340 nm

Reference Range: 6-37 U/L

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase

Catalyze the hydrolysis of

phosphomonoesters

Requires Mg2+ activator

Evaluation of hepatobiliary and bone

disorders.

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase

Assay for Enzyme Activity

Bowers and McComb

Based on molar absorptivity of pNitrophenol

Absorbance is measured at 405 nm

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase (ALP)

Methods
1-4. Bodansky,
Shinowara,
Jones, Reinhart
5.
Bessy, Lowry &
Brock
6.
Bowers &
McComb
7.
King and

Substrate

End
Product
Inorganic
-glycero-phosphate PO4
+
Glycerol
p-nitrophenyl
phosphate

pnitrophenol
(yellow)

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase (ALP)

Reference Range

30 90 U/L (adult)

70 220 U/L (0 3 months)

50 260 U/L (3 - 10 years)

60 295 U/L (10 - puberty)

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase (ALP)

ALP Isoenzymes

Differentiation by electrophoresis
1.
Liver ALP
2.
Bone ALP
3.
Placental ALP
4.
Intestinal ALP

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase (ALP)

ALP Isoenzymes
1.
Liver ALP
Fastest isoenzyme and in liver
diseases
Fractions: Major liver and fast liver ( 1)
band
2.
Bone ALP
Heat labile fraction
in bone disease, healing of bone

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase (ALP)

ALP Isoenzymes
3.
Placental ALP
Most heat stable fraction and in pregnancy
4.
Intestinal ALP
Slowest moving fraction, in blood groups B or
O
in fatty meal and GIT disorders
Note: Placental and Intestinal ALP are inhibited
by phenylalanine (chemical inhibition)

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase (ALP)

ALP Isoenzymes

Differentiation by Heat Stability

Serum is heated at 56C for 10

minutes
1. Liver ALP
ALP residual activity is to >20%
2. Bone ALP
ALP residual activity is to <20%

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
2. Alkaline Phosphatase

Significance of Isoenzyme fractions

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
4. Gamma-Glutamyltransferase (GGT)

Catalyze the transfer of the -glutamyl

residue from -glutamyl peptides to
amino acids, H20.

Diagnosis hepatobiliary disorders and

chronic alcoholism

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
4. Gamma-Glutamyltransferase (GGT)

Assay for Enzyme Activity

Szaz Assay

Absorbance of p-Nitroaniline is
measured at 405-420 nm

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
1. Amylase (AMS)

Catalyzes the breakdown of starch and

glycogen via , 1-6 branching linkages

Increased in acute pancreatitis

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
1. Amylase (AMS)

Assay for Enzyme Activity

Amylase Methodologies

1.
2.
3.
4.

Amyloclastic
Chromogenic
Saccharogenic
Continuous monitoring

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
1. Amylase (AMS)

Assay for Enzyme Activity

Amylase Methodologies
Measures the disappearance of starch
1.
Amyloclastic substrate
Starch-iodine comp. (dark-blue) color
intensity
Measures the appearance of the product
2.
Saccharogeni
c

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
1. Amylase (AMS)

Assay for Enzyme Activity

Amylase Methodologies
Measures the in color
3.
Chromogeni Insoluble starch-dye soluble starch-dye
c
fragments
4.
Coupling of several enzyme systems to
Continuous
monitor amylase activity

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
1. Amylase (AMS)

Assay for Enzyme Activity

in absorbance at 340nm is measured

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
1. Amylase (AMS)

Amylase Isoenzymes
i.
Salivary Amylase ptyalin (fast
moving)
ii.
Pancreatic Amylase amylopsin (slow
moving)

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
2. Lipase (LPS)

Hydrolyzes of fats to produce alcohols

and FA

Earliest and specific marker for acute

pancreatitis

Larger molecule, remains in circulation

(7 days)

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
2. Lipase (LPS)

Enzymes
V.

Enzymes of Clinical Significance

C.

Pancreatic Enzymes
2. Lipase (LPS)

Assay for Enzyme Activity

1. Cherry
Crandall

Substrate
Titrating
agent
Indicator
Endpoint

2. Tietz

50% olive oil (triolein)

0.4N NaOH
Thymolpthalein
Phenolpthalein
+ Veronal
Fatty Acid (Oleic Acid)

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
1. Acid Phosphatase (ACP)

Catalyze the hydrolysis of

phosphomonoesters

Evaluation of metastatic carcinoma of

prostate.

Forensic investigation of rape

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
1. Acid Phosphatase (ACP)

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
1. Acid Phosphatase (ACP)

Phosphatase inhibitors
i.
L-tartrate ions

inhibits specific prostatic ACP

Total ACP ACP after inhibition =
prostatic ACP
ii.
Formaldehyde and Cupric ions

inhibits red cell ACP

Enzymes
V.

Enzymes of Clinical Significance

B.

Liver Enzymes
1. Acid Phosphatase (ACP)

Assay for Enzyme Activity

Reference Range: Prostatic ACP: 0 -3.5

ng/ml

Methods
1. Quantitative end
point

Substrate
Thymolpthalein
monophosphate