Enzymes

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.

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.

A.
MI Profile
Enzymes
V.

A.
MI Profile
Methods of Determination
i.
Forward Reaction (Tanzer-Givarg)
ii.
Reverse Reaction (Oliver-Rosalki)
Enzymes
V.

A.
MI Profile
i.
Measure in absorbance at 340 nm
Optimum pH is 9.0
Enzymes
V.

A.
MI Profile
i.
Coupled-enzyme
assay
Auxiliary
enzyme
Indicator
enzyme
Enzymes
V.

A.
MI Profile
ii.
in absorbance at 340 nm is
determined
6x faster than forward reaction
Optimum pH: 6.8
Enzymes
V.

A.
MI Profile
ii.
Enzymes
V.

A.
MI Profile
i.
ii.
Enzymes
V.

A.
MI Profile
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.

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.

MI Profile
A.
1.
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.

MI Profile
A.
1.
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.

MI Profile
A.
1.
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.

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.

A.
MI Profile
Enzymes
V.

A.
MI Profile
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.

A.
MI Profile
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.

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.

A.
MI Profile
Assay of Enzyme Activity

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

A.
MI Profile

a.
Wacker method
Forward Reaction (Lactate Pyruvate)

in absorbance is monitored at 340 nm
Optimal pH is 8.3 8.9
Enzymes
V.

A.
MI Profile

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.

A.
MI Profile
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.
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.

A.
MI Profile
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
Significance of Isoenzyme
fractions
Enzymes
V.

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.

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.

B.
Liver Enzymes
Enzymes
V.

B.
Liver Enzymes
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.

B.
Liver Enzymes
Uses Lactate Dehydrogenase and

monitors decrease in absorbance at
340 nm
Reference Range: 6-37 U/L
Enzymes
V.

B.
Liver Enzymes
2. Alkaline Phosphatase
Catalyze the hydrolysis of

phosphomonoesters
Requires Mg2+ activator
Evaluation of hepatobiliary and bone

disorders.
Enzymes
V.

B.
Liver Enzymes
Enzymes
V.

B.
Liver Enzymes
Bowers and McComb
Based on molar absorptivity of pNitrophenol
Absorbance is measured at 405 nm
Enzymes
V.

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.

B.
Liver Enzymes
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.

B.
Liver Enzymes
ALP Isoenzymes
Differentiation by electrophoresis
1.
Liver ALP
2.
Bone ALP
3.
Placental ALP
4.
Intestinal ALP
Enzymes
V.

B.
Liver Enzymes
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.

B.
Liver Enzymes
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.

B.
Liver Enzymes
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.

B.
Liver Enzymes
Significance of Isoenzyme fractions
Enzymes
V.

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.

B.
Liver Enzymes
4. Gamma-Glutamyltransferase (GGT)
Szaz Assay
Absorbance of p-Nitroaniline is
measured at 405-420 nm
Enzymes
V.

C.
Pancreatic Enzymes
1. Amylase (AMS)
Catalyzes the breakdown of starch and

glycogen via , 1-6 branching linkages
Increased in acute pancreatitis
Enzymes
V.

C.
Pancreatic Enzymes
1. Amylase (AMS)
Amylase Methodologies
1.
2.
3.
4.
Amyloclastic
Chromogenic
Saccharogenic
Continuous monitoring
Enzymes
V.

C.
Pancreatic Enzymes
1. Amylase (AMS)
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.

C.
Pancreatic Enzymes
1. Amylase (AMS)
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.

C.
Pancreatic Enzymes
1. Amylase (AMS)
in absorbance at 340nm is measured
Enzymes
V.

C.
Pancreatic Enzymes
1. Amylase (AMS)
Amylase Isoenzymes
i.
Salivary Amylase ptyalin (fast
moving)
ii.
Pancreatic Amylase amylopsin (slow
moving)
Enzymes
V.

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.

C.
Pancreatic Enzymes
2. Lipase (LPS)
Enzymes
V.

C.
Pancreatic Enzymes
2. Lipase (LPS)

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.

B.
Liver Enzymes
1. Acid Phosphatase (ACP)
Catalyze the hydrolysis of

phosphomonoesters
Evaluation of metastatic carcinoma of

prostate.
Forensic investigation of rape
Enzymes
V.

B.
Liver Enzymes
Enzymes
V.

B.
Liver Enzymes
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.

B.
Liver Enzymes
Reference Range: Prostatic ACP: 0 -3.5

ng/ml
Methods
1. Quantitative end
point
Substrate
Thymolpthalein
monophosphate

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Chapter Outline

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Storage of high-energy creatine

Highest activities in skeletal muscle,

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Enzymes of Clinical Significance

Hemolysis cause false CK due to AK

Physical activity and IM injections

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

CK-MB: <6% of total CK

Enzymes of Clinical Significance

Creatinine Kinase (CK)

Enzymes of Clinical Significance

Creatinine Kinase (CK)

Diagnostic Significance of CK Isoenzymes

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

Enzymes of Clinical Significance

Creatinine Kinase (CK)

Enzymes of Clinical Significance

Creatinine Kinase (CK)

Enzymes of Clinical Significance

Serum glutamic-oxaloacetic transaminase

Transfer of amino group in aspartate and keto acids

Enzymes of Clinical Significance

Enzymes of Clinical Significance

AST levels begin to rise in 6-8 hours,

Also in hepatocellular and skeletal

Enzymes of Clinical Significance

Assay for Enzyme Activity

Uses malate dehydrogenase and

Falsely in hemolyzed sample

Reference Range: 5 30 U/L

Enzymes of Clinical Significance

Interconversion of lactate and pyruvate

Widely distributed, highest activities in

Enzymes of Clinical Significance

Assay of Enzyme Activity

Enzymes of Clinical Significance

Assay of Enzyme Activity

Forward Reaction (Lactate Pyruvate)

Enzymes of Clinical Significance

Assay of Enzyme Activity

Reverse Reaction (Pyruvate Lactate)

Enzymes of Clinical Significance

LD begin to rise within 10-24 hrs,

Reference Range: 100-225 U/L

Enzymes of Clinical Significance

Tetramer containing two active sub-units

Enzymes of Clinical Significance

Relative concentration in normal

In AMI and intravascular hemolysis,

Enzymes of Clinical Significance