Chapter 2 enzymology.ppt

Learning Objectives for the Chapter
Upon completion of this chapter the student will be able
to:
 Describe the chemical makeup, general
characteristics, classes and nomenclature of enzymes.
 Discuss how enzymes act as catalysts in specific
biological reactions, in terms of activation energy and
general nature
2

Learning Objectives for the Chapter
to:
 Explain plasma specific versus non-plasma specific
enzymes, factors that affect the rate of enzymatic reactions,
including cofactors, coenzymes and inhibitors.
 Describe enzyme kinetics, fixed time assay and continuous
monitoring assay, the unit of enzyme activity and the
calculation for activity and volume activity.
3

Learning Objectives for Chapter
to:
• Discuss the biochemical characteristics, source,
clinical significance, methods of analysis,
interpretation of results and sources of errors for
selected enzyme tests:
• Transferases
• Phosphatases
• LD, CK
• Amylase and Lipase
4

Learning Objectives for
this Lesson
Upon completion of this lecture the student will be able
to:
 Describe the chemical makeup and general
characteristics of enzymes.
 Discuss the 6 main classes of enzymes in terms of
general function, listing some common examples.
 Explain the nomenclature for enzymes listing some
common examples.
5

Learning Objectives for
this Lesson
 Discuss how enzymes act as catalysts in specific
biological reactions.
 Compare activation energy in a catalyzed versus a non-
catalyzed chemical reaction.
 Explain the general relationship between enzyme,
substrate and product; nature of enzymes in chemical
reaction
6

Learning Objectives for this lesson:
 Explain plasma specific versus non-plasma specific
enzymes
 Describe factors that affect the rate of enzymatic
reactions.
 Discuss the role of cofactors with enzymes
7

Learning Objectives for this lesson:
 Define enzyme kinetics, fixed time assay and
continuous monitoring assay
 On a Michaelis-Menten curve, identify where a
reaction proceeds in first-order kinetics and zero-
order kinetics
 Recognize a Lineweaver-Burk transformation and
explain why it is useful in describing enzyme reaction
velocity
 Describe three kinds of inhibitors on enzyme reaction
velocity
8

Learning Objectives
Upon completion of this lecture the student will be able:
 Compare fixed-time and continuous monitoring
kinetic assays of enzyme activity
 Identify the unit used to report enzyme activity.
 Calculate enzyme activity (volume activity)
9

Outline
 Diagnostic Enzymology
 Introduction (enzymology from a clinical point of view)
 Classification and Nomenclature of enzymes
 Mechanism of enzymes action
 Nature of enzymes regarding energy requirements of
chemical reaction
 Enzyme kinetics (substrate concentration, temperature,
cofactors, coenzymes, inhibitors, pH)
 Enzyme Assay Techniques
10

Outline
 Fixed time (fixed time kinetic) assay techniques
 Continuous (kinetic) monitoring assay techniques
 Plasma specific versus non- plasma specific enzymes
 Factors affecting enzyme level in plasma or serum
11

Outline
 Selected Enzyme Tests
 The transferases (AST, ALT, GGT)
 The phosphatases
 Lactate dehydrogenase
 Creatine kinase
 Amylase
 Lipase
 Principles & techniques for enzyme determination
 Calculation of enzyme activity (volume activity)
 Clinical significance, reporting, documentation and
interpretation of enzyme results
12

Introduction to Enzymology
 Used for diagnosis and treatment of diseases
 Enzymes are protein catalysts
 Enzymes are present in small quantities in body fluids
 Enzymes are measured by “what they do”
13

Enzyme Chemical Makeup
Enzymes are proteins
 Protein structures are composed of :
 Primary bonds
 Secondary bonds
 Tertiary bonds
 Quaternary bonds
 Conjugated with carbohydrates or other compounds.
14

Enzyme Characteristics
 Primary structure allows for ionization.
 Tertiary and quaternary structure of enzymes produces
active sites for substrate binding.
15

Properties of Enzymes
 Temperature dependent activity
 Easily denatured
 Coenzyme and metal activators
 Coenzymes are organic molecules that assist enzymes in
conversion of substrate to product by contributing H+ or
other necessary conditions
 Metal activators contribute to the ionic activity of the
enzyme. Examples of activators include Cl or Mg
16

Classes of Enzymes:
6. classes of enzymes
1. Oxido-reductase (oxidation- reduction reaction between two
substrates)
2. Transferase(transfer of a group other than hydrogen
from one substrate to another)
3. Hydrolase (catalyze hydrolysis of an ether, ester,etc)
4. Lyase (the removal of groups from substrates without
hydrolysis)
5. Isomerase (interconversion of geometric, optical, or
positional isomers )
6. Ligase[Synthetases] (joining (synthesis) of two
substrate molecules, )
 Name describes type of reaction involved 17

Nomenclature of Enzymes
 Arbitrary in the past
 Suffix -ase
 Reaction named
 Combination (trivial, common and semi-systemic)
 Standardized system of names was recognized
18

Enzyme Nomenclature
 Enzyme Commission of the International Union of
Biochemistry
 unique numerical names consisting of four numbers
separated by periods to indicate class, subclass, sub-
subclass and a specific serial number.
 Lactate dehydrogenase, LD, EC 1.1.1.27
 Alanine transaminase, ALT, formerly serum glutamate
pyruvate transaminase, SGPT, EC 2.6.1.2
19

Enzyme Nomenclature
 2 Names for each enzyme
 Systematic name: the reactions catalyzed, associated
with a unique numerical code designation
 Recommended, trivial or practical name: a
simplification , suitable for everyday use.
20

Mechanism of Enzyme Action
 This equation represents an enzymatic reaction:
 E+S ↔ ES → P+E
 E = enzyme, S = substrate, P = product
 Formation of the ES complex occurs rapidly.
 2 Models for specific binding of substrate to enzyme
 Lock and key specificity (Fisher’s)
 Induced Fit Model
 After binding, enzyme takes a shape complimentary to
substrate
21

Nature of Enzymes
 Substrate Specificity
 Absolute specific enzymes;
 Stereo-isomeric specific enzymes
22

Nature of Enzymes
 Group specific enzymes: broader specificity and act
on a group of related substrates rather than on a
single substrate. Eg, the phosphates that split
phosphate from a group of a large variety of organic
phosphate esters.
 Bond specific enzymes (function-specific): These
are enzymes with low specificity; they act on
substrates containing a particular functional group or
chemical bond. Eg. peptidases, esterases,
amidases.
23

Energetics of Catalyzed Chemical
Reactions
Enzymes:
 Act as catalysts in most physiological reactions
 Lower the activation energy of the substrate
(or reactants) so a reaction can take place.
 Do not change the equilibrium constant of the
reaction.
 Do change rate at which equilibrium is established.
24

Catalysts reduce the free or “activation” energy
required to activate a chemical reaction
Activation energy for
non-catalyzed reaction
Activation energy for
catalyzed reaction
Reaction
Free
energy
Initial reaction
state
Equilibrium
Drawn by John Wentz, MS,CLS
25

Enzyme Activity
Review Question regarding mechanism of action:
 What is a product?
Answer: compound forming from the
substrate in the chemical reaction.
S+E S-E P + E
26

Enzyme Activity Review Question:
What is the type of protein that accelerates the speed of
a chemical reaction by binding specifically to a
substrate forming a complex, lowering the activation
energy in the reaction without becoming consumed or
without changing the equilibrium of the reaction?
Answer: Enzyme
27

2.1. Introduction to Enzyme Kinetics
 Definition of Enzyme Kinetics:
 The study of the rate of enzyme reactions.
 Factors affecting enzyme kinetics
 Enzyme concentration
 Substrate concentration
 Product concentration
 pH and ionic strength
 Temperature
 Cofactors and Inhibitors
28

Effects of Enzyme Concentration
on Rate
Ef + S ES Ef+P
 If substrate concentration exceeds enzyme
concentration, rate is proportional to enzyme activity.
 The basis of clinical assays: excess substrate available
in reagent and unknown concentration of enzyme in
serum.
 ↑ enzyme activity = ↑ rate
29

Large Amounts of Enzyme Activity
 When substrate is depleted from a high rate of product
formation, zero order kinetics is no longer observed.
 Activity needs to be determined by either:
 Diluted sample
 Decreased ratio of sample to reagent
 Fast kinetics
 Final activity is determined by a dilution factor.
30

Enzyme Activity
 Coupled enzymatic reactions are linked.
 1st enzyme catalyzes a primary reaction
 2nd enzyme catalyzes a secondary reaction
 In vitro coupled reactions:
 secondary enzyme
 provided in the reagent
 produce product
 indicating reaction.
 Secondary enzyme = indicating enzyme
31

Measuring an Analyte Using an Enzyme
 Enzymes can be used to measure an analyte with high
level of specificity.
E.g. Ammonia analysis:
NH4
+ + 2-oxoglutarate + NADPH ----GLDH ------>glutamate
+ NADP +H2O
 Only two absorbance readings are taken
 A decrease in absorbance is measured at 340 nm due
to the formation of NADP at 37 0C
32

Effect of Substrate on Reaction Rate
 Reaction rate increases proportionately with an
increase in substrate concentration, [S].
 Defined as first-order kinetics.
 Km is a constant specific for each enzyme:
the [S] that corresponds to ½ maximum velocity.
 [S] increases until available enzyme is saturated and
reaction velocity flattens out or plateaus. Rate does
not change with added substrate.
 Defined as zero-order kinetics.
33

Michaelis-Menten Curve
15
10
10
30
20
Substrate Concentration = [S]
Km ≈ 4
V max = maximum velocity
(Reaction follows zero-
order kinetics).
½ maximum velocity
(Reaction follows first-order
kinetics)
Drawn by John Wentz, MS, CLS
Reaction
Velocity
(v)
34

The Lineweaver-Burk
Transformation
 Determining Vmax using
Michaelis-Menten curve is
difficult.
 Lineweaver-Burk
transformation is easier
because it yields a straight
line plot.
 1/v = [Km/Vmax] x 1/[S] +
1/Vmax
1
V
-1
Km
1
V max
1
[S]
Drawn by John Wentz, MS,CLS
35

Effect of Product on Reaction Rate
 Accumulated product may inhibit reaction rate.
 Mass action effect
 Inhibition
 Changes pH
36

Effect of pH and Ionic Strength on Rate
 Enzymes are proteins.
 Proteins change shape or net molecular charge as pH
changes.
 Most enzymes only work in pH 7.0-8.0
 In-vitro diagnostics (clinical assays) - buffers used to
control pH.
37

Effect of Temperature
 Chemical reactions rates are increased by increasing
temperature, including enzyme reactions up to optimum
temperature.
 At 40° – 50° C most enzymes are inactivated.
 At 60° – 70° C denatured irreversibly.
 Colder temps.(i.e., 4°C) reversibly inactivate;
storage temp of samples if analysis is to be delayed.
38

Importance of Temperature
 37°C is ideal for most enzymatic reactions, but some
procedures use 35° or 30° C
 Temperature of rate reactions must be tightly
controlled (± 0.1 ° C). Use of water-bath or other
temperature controlled equipment is necessary.
39

Some Enzyme Reactions Require
Cofactors (Activators)
 Non-protein cofactors:
Cations: Ca 2+,Fe 2+, Mg 2+, Mn 2+, K +, Zn 2+;
Anions: Cl -, Br–
 Alters enzyme configuration to promote binding or
enable binding site. Increases enzyme activity.
 Some of these ions are tightly bound to enzyme
molecule, others transiently.
40

Some Enzyme Reactions Require
Coenzymes
 Coenzymes - class of molecules necessary for the
enzyme to catalyze
 eg. prosthetic group such as NAD/NADP, vitamins
 Apoenzyme + Coenzyme  Holoenzyme
41

Inhibitors Interfere with Enzyme
Reactions
 Affect Vmax and Km of enzymatic reactions.
 Three types of inhibitors:
1. Competitive inhibitors.
2. Noncompetitive inhibitors.
3. Uncompetitive inhibitors
42

Competitive Inhibitors
 Compete with the substrate for the active site of the
enzyme
 prevent formation of product
 have a higher Km than the preferred substrate
 can be overcome by addition of more substrate
Eg: Lactate and a-dehydroxybutyrate for LD
43

Noncompetitive Inhibitors
 Bind on allosteric site but not the active sites of
enzyme
 Can not be overcome by addition of more substrate
 Prevents formation of product despite the enzyme-
substrate complex.
44

Uncompetitive Inhibitors
 Bind to the enzyme-substrate complex
 Prevent the formation of product
 Not overcome by addition of substrate
45

Enzyme Assay Techniques
2 main types of assay techniques:
 Fixed time kinetic assay techniques
 Continuous (kinetic) monitoring assay
techniques
46

Fixed-Time (or 2- point) Assays
 Substrate is added and Abs is measured after a
predetermined interval.
 Does not indicate substrate depletion or presence of
inhibitors in reaction system.
 Fixed-time assays are best for batch runs (multiple
samples ran simultaneously)
 If enzyme activity is very high, substrate is depleted
too quickly.
47

Continuous (kinetic) monitoring assay
techniques
 This is also multi-point
 Abs measurements made at specific intervals
 usually 30 to 60 sec
 Continuous Monitoring refer to a recording
spectrophotometer taking more frequent
measurements
48

Determining Enzyme Kinetic Activity
49

Example of Continuous monitoring: ALT
method
Amino acid + substrate –(enzyme, coenzyme)amino
acid + product
Substrate (product of 1st) + coenzyme -(2nd enzyme)
product + reduced coenzyme
 Coupled reaction at 37 0 C
 Decrease in Abs. 340 nm (continuous monitoring).
50

Example Enzyme Reaction
L-Alanine + 2-oxoglutarate -- ALT- Glutamate +
Pyruvate
NADH + H+ + Pyruvate -- LDH  Lactate + NAD+
 Absorbance due to NAD can be measured at 340 nm
 The molar absorptivity (epsilon) of NAD at 340 nm
is 6220 cm. L/ mole
 Refer to next slides for results
51

Enzyme Kinetic Assay
 ΔAbs is determine as Absorbance at time 1 subtracted
from Absorbance at time 2
 ΔAbs = A2 – A1
 Sometimes Absorbance decreases with time so A2- A1
is a negative number.
 International standards have this number indicated as
negative and is multiplied by a negative activity factor
so the final activity is still a positive number.
52

Example of a Kinetic
Assay – Continued
Time Abs ΔAbs
0 sec .0450
10 .0410 -0.004
20 .0380 -0.003
30 .0330 -0.005
40 .0285 -0.004
50 .0255 -0.003
60 .0235 -0.002
ΔAbs = -0.021/min
Temperature dependent
53

Decreasing Absorbance Per Minute
 In ALT the absorbance decreases over time
 The result is -A X F
Min
 Where F340 = -1746
 So final activity is a positive number U/L
 ALT activity would be -0.021 x – 1746 = 37 IU/L
 This results is within the reference range for this
method: 37 IU/L (reference range is 6-37 IU/L)
54

Plasma specific versus non- plasma
specific enzymes
 Plasma specific enzymes have function in plasma
 Produced in liver but secreted into plasma
 Clotting factors
 Non-plasma specific enzymes are found in cells
 Produced in specific cells
 Release into plasma during disease
 Amylase, ALT, LD
55

Factors affecting enzyme level in
plasma or serum
The factors affecting enzyme levels in plasma are:
 Rate of enzyme release from cells
 Extracellular Fluid volume of distribution of the
enzyme
 Enzyme removal rate from plasma (catabolism or
excretion)
 Plasma factors which may affect the method of assay
(inhibitors or activators)
56

Principles of Enzyme Determination
 Enzyme concentration in serum is not clinically
significant.
 Enzyme recently released from diseased or dying cells
is significant.
 The amount of functional (activity of) enzyme is
significant.
 Enzyme activity is standardized as International units
of activity = IU.
57

Principle of Enzyme Activity
Determination
 Enzyme activity is measured, since enzyme
concentrations are not clinically significant.
 Some enzyme assays measure the reduction or oxidate
of coenzymes NAD to NADH (or NADH to NAD)
photometrically at 340 nm.
 Enzyme activity is measured when rate is constant, or
zero-order kinetics.
 Temperature, pH, ionic strength must be maintained.
58

Enzyme Activity Determination
 Kinetic methods (continuous monitoring)
 Absorbance (Abs) measured at regular intervals
(e.g., 10 or 30 seconds)
 Measurements begin after lag phase
 If there is a fluctuation in temperature, volume,
improper timing, the  absorbance should not be
calculated
 The reaction should be investigated
 The problem should be solved
59

Enzyme Activity Determination
 Measurements continue until little or no change in
Abs between measurements (substrate depleted).
 Average change in Abs (Δ Abs)/ minute is calculated.
60

Calculating and Reporting Enzyme
Activity/Volume Activity
 Enzyme activity is reported as International Units/ liter
(IU/L) calculated from  Abs/ min x molar absorptivity
(epsilon) of the product in cm.L/ mol x conversion factor
for volume x ratio of total volume / sample volume in mL.
 Commonly determined as change in  Abs/ min x Factor.
 Review the results again on the next slides
61

Example of Problems with a Kinetic
Assay
Time Abs ΔAbs
0 sec .0450
60 .0410 -0.004
120 .0380 -0.003
180 .0390 +0.001
240 .0285 -0.0105
300 .0255 -0.003
360 .0235 -0.002
ΔAbs =
Notice the
absorbance readings
are fluctuating here.
62

International Units of Enzyme Activity
and Volume Activity
 IU = the amount of enzyme needed to convert 1
micromole of substrate to product per minute.
 Volume activity = IU/L
63

Summary: Enzyme Kinetics
 Enzymes act as catalysts by lowering the activation
energy required for a reaction to take place.
 The action of enzymes is summarized in the
formula:
E+S ↔ ES → E + P
64

Question for You:
The equilibrium coefficient (Km) that represents the
likelihood of a particular enzyme-substrate complex to
dissociate and form product is determined from the
Michaelis-Menten curve as_________
Answer: ½ V max
65

Summary: Enzyme Kinetics – Continued
 Michaelis-Menten curve describes constant Km, the
substrate conc. that corresponds to ½ V max – the
maximum reaction velocity or rate.
 Lineweaver-Burk transformation gives inverse: 1/Vmax
and 1/Km
 But yields a linear line instead of a hyperbolic curve.
66

Question for You:
Unexpected low activity results are found for a sample of
known LD activity. The substrate of choice is lactate but a-
dehydroxybutyrate is found to be present in the reagent. When
excess lactate is added to the reagent mix, the observed activity
in the known sample is higher and meets expectations. Adding
the additional lactate describes overcoming _______ inhibition.
Answer: Competitive
67

 The reaction rate increases with substrate concentration
in first-order kinetics. The rate stabilizes when there is
an excess of substrate – zero-order kinetics.
 Some enzymes require cofactors or activators, often
metallic ions, smaller proteins or vitamins.
68

Question for You:
The D Abs/min in lactate dehydrogenase analysis was
found to be 0.010 Abs/min. The activity factor (F) based
on molar absorptivity and ratio of sample to total volume
is 4800. What is the LD activity of this sample in IU/L?
Answer: 0.010 x 4800 = 48 IU/L
69

 Enzyme inhibitors slow or
stop reaction rate. Three
types: Competitive,
Noncompetitive, and
Uncompetitive.
 Enzyme assays are
designed in zero-order
kinetics (constant rate).
Many clinical assays use
fixed-time (end-point)
methodology.
70

 Temperature, pH and ionic strength must be tightly
controlled in enzymatic reactions.
 Enzymes exist in very small concentrations in body
fluids.
 Enzyme activity is measured and reported.
 The standard unit of enzyme activity: IU/L
71

Introduction to Transferases
 Catalyze interconversion of functional groups.
 Transaminase- Aminoacids to a-oxoacids
 - insert for GGT
o Names:
AST; formerly Glutamate Oxaloacetate
transaminase, GOT
ALT; formerly Glutamate Pyruvate Transaminase, GPT
73

(ALT) Biochemical
Theory & Metabolic
Pathways
Transaminase - transfers amino groups
 Other descriptive names:
 serum glutamic pyruvate transaminase (SGPT)
 alanine aminotransferase ( ALAT)
 ALT catalyzes the reaction:
L-Alanine + a-Oxoglutarate  Pyruvate + L-
Glutamate
74

Clinical Significance of
ALT and AST
Damage to tissue can release different types of enzymes
based on their location.
 Mild inflammation of the liver
 Reversibly increases the permeability of the cell membrane
 Releases cytoplasmic enzymes: AST
 Necrosis releases mitochondrial ALT, AST
75

Clinical Significance of ALT
 Hepatocellular necrosis releases mitochondrial ALT
 Associated with liver inflammation (hepatitis)
 Drugs overdose or toxicity
 Infections from Viruses or bacteria
 Alcohol
76

Analytical Methodology of ALT
Continuous Monitoring Method
 l-Alanine + a-oxoglutarate -(ALT, P-5’-P)l-glutamate
+ pyruvate
 Pyruvate + NADH + H+ –(LD)-> lactate + NAD+
 Decrease in Abs. 340 nm (multi-point analysis).
77

Calculating Absorbance
Per Minute
 In ALT the absorbance decreases over time
Min
 Where F340 = -1746
78

Continuous Monitoring Analysis
Results Example
 The following results for ALT were determined:
 Are the results progressing in the expected direction?
Time (min) Absorbance
0 1.350
1 1.300
2 1.250
3 1.201
Answer: yes, they are decreasing
79

Continuous Monitoring Results
of ALT analysis
What is the  Abs for each minute?
Answer: -0.050/min; -0.050/min; -0.049 /min
Are the results consistent?
Answer: Yes, absorbance decreases
consistently
Average = -0.050 /min
80

Fixed End-Point Assay for ALT
 Photometric Method
 ALT is coupled with 2,4 dintrophenylhydrazine 
dintrophenylhydrazone (chromogenic product)
 Product measured photometrically
81

Specimen for ALT
 Nonhemolyzed serum or plasma.
 Heparinized plasma
 < 2 day old samples
 Fasting specimen is preferred
82

Reference Values of ALT
Serum or plasma reference ranges vary with method
 Reference ranges reflect the normal amount in serum
or plasma:
 Adult male <45 U/L
 Adult female <34 U/L
83

Quality Control
 A normal & abnormal quality control sample should
be analyzed along with patient samples, using
Westgard or other quality control rules for acceptance
or rejection of the analytical run.
 Assayed known samples
 Commercially manufactured (Humastar)
 Validate patient results
 Detects analytical errors.
84

Sources of Error in ALT
 Nonlinear results from side reactions can be repeated
with diluted sample
 Hemolyzed samples cause false positive
 Loss of activity if specimen is stored at room
temperature
 Unstable analytical temperature (deviation from 37 0 C
 Unstable photometer
 Substrate exhaustion due to high levels of enzyme
activity
85

Transaminase (AST)
Biochemical Theory &
Metabolic Pathway
Serum glutamic oxaloacetic transaminase (SGOT) or
aspartate aminotransferase (ASAT/AAT)
 Transfers amino groups to form oxaloacetate
 Found in serum from various tissues
 Associated with hepatocytes
86

Clinical Significance of AST
 Hepatocellular inflammation releases cytoplasmic AST
 Hepatocellular necrosis releases mitochondrial AST.
 Associated with liver inflammation (hepatitis)
 Drugs overdose or toxicity
 Infections from Viruses or bacteria
 Alcohol
 Other organ diseases
 Myocardia infarction
87

Analytical Methodology of AST
 Amino acid + substrate -(AST, coenzyme)amino
acid + product
 Substrate (product of 1st) + coenzyme -(2nd enzyme)
product + reduced coenzyme
 Decrease in Abs. 340 nm (continuous monitoring).
88

Analytical Methodology of AST
 Continuous Monitoring Method
 l-Aspartate + a-oxoglutarate -(AST, P-5’-P)l-
glutamate + oxaloacetate
 Oxaloacetate + NADH + H+ –(MDH)-> l-malate +
NAD+
 Decrease in Abs. 340 nm (multi-point assay)
89

Per Minute
 In AST the absorbance decreases over time
Min
 Where F340 = -value
 N.B. Factor is dependent on specific methodology.
90

Reference Ranges of
AST
Serum or plasma reference ranges vary with method
 Reference range:
 Adult male <35 U/L
91

Interpretation of
Transaminases
HIV treatment, especially the reverse transcriptase
inhibitors are associated with metabolic
complications:
 Pancreatitis, hypertriglyceridemia, and lactic acidosis
 Hepatomegaly, and hepatic inflammation
Patients taking these medications will have liver enzyme
levels monitored every few months to monitor for
these complications
92

Here is a question for you:
List the initial substrate and the final product(s) for
AST.
Answer: initial substrate = a-
oxoglutarate and final products=
maltate + NAD+
93

Fixed End-Point Assay for AST
 Photometric Method
 AST is coupled with 2,4 dintrophenylhydrazine 
dintrophenylhydrazone (chromogenic product)
 Product measured photometrically
94

Specimen for AST
 Nonfasting may falsely increase
95

Quality Control
96

Analytical Errors for
ASTActivity:
Sources of errors in testing for AST:
 Unstable temperature (deviation from 37 0 C
 Substrate exhaustion due to high levels of enzyme
activity
97

Sources of Error in AST
 Presence of ammonia will consume the NADH
with diluted sample
 Hemolyzed specimens cause false increase
 Nonfasting specimens may falsely increase
 Loss of activity if stored at room
temperature for > a day
98

Transferase (GGT)
Biochemical Theory &
Metabolic Pathway
 Involved in the transfer of glutamyl group
 Glutathione metabolism
 Cysteine product preserves intracellular homeostasis
of oxidative stress
99

Clinical Significance of GGT
 Found in biliary ducts of liver, kidney tubules, and
prostate
 Associated with disease in the liver such as
hepatobiliary obstruction or inflammation
 Elevated because of alcoholism
100

Specimen for GGT
 Nonhemolyzed serum
 EDTA plasma
101

Analytical Methods for GGT
I. Serum Start Method:
 Peptide GGPNA substrate – GGT  p-
nitroaniline
 Increased in Abs at 405 nm
102

Analytical Methodology: GGT
by
II. Continuous Monitoring method/Substrate Start
Method
 Coupled reaction at 37 0C
 Gamma-glutamyl-p-nitroanalide + (HCl) glycylglycine
–(GGT, pH 8.2)--> Gamma-glutamylgylcylglycine + p-
nitroaniline.
 Measure increase Abs. 405 nm as determined by
continuous or 2- point monitoring with a manual or
automated spectrophotometer
103

Per Minute
 In GGT the absorbance inreases over time
 The result is A X F
Min
 Where F405 = value
104

Interpretation of GGT
Serum or plasma reference ranges vary with method.
Reference range
 Adult Male <55 U/L
Serum levels increase in hepatobiliary obstruction or
inflammation or in alcoholism
105

Quality Control
106

Sources of Errors for
GGT Activity:
 Loss of activity if stored at room temperature for
longer than a day
 Heparinized plasma produces turbid samples
 Other anticoagulants( citrate, oxalate, fluoride)
depress activity of enzyme
107

Pre-analytical Errors for GGT
 Hemolysis
 Samples > 2 days old not stored in refrigerator or
freezer.
108

Analytical Errors for GGT
 Substrate exhaustion from extremely elevated enzyme
activity
 Unstable temperature during analysis
109

Automated analysis of
Transferase Enzymes
110

Transferase Enzyme
Analysis Results
 Before Reporting:
 Check that quality control samples are accepted.
 Check that results correlate well with other results
 Avoid these mistakes in reporting:
 Wrong name
 Incorrect units or reference range
 Reference range not matched to gender
 Transcribing wrong number
 Report too late
111

Documentation of
Transferase Enzyme
Analysis
 Record patient results in result logbook
 Record QC results in QC logbook
 Retain records for recommended time
112

Learning Objectives
After listening to the lectures and completing the
exercises, the student will be able to:
 Describe the biochemical theory & metabolic pathways,
and physicochemical properties of alkaline phosphatase
(ALP) and acid phosphatase (ACP)
 Discuss the normal & abnormal states affecting levels of
ALP and ACP
 Describe the principles of alkaline phosphatase, analysis in
terms of key reagents and their role.
114

Learning Objectives
 Describe the principles of acid phosphatase, analysis in
 Differentiate causes of common preanalytical, analytical
and postanalytical errors in alkaline phosphatase and acid
phosphatase analysis.
 Interpret results of ALP and ACP compared to reference
ranges.
115

Outline of Phosphatase Lecture
 Introduction
 Source
 Clinical Significance
 Methods of Analysis including Calculations
 Specimen
 Interpretation of Results
 Quality Control
 Sources of Errors
 Reporting and Documentation
 Summary
116

Outline of Lecture: ALP, ALP
Isoenzymes and ACP
 Introduction
 Source
 Methods of Analysis
 Calculations
 Specimens
 Quality Control
 Summary 117

Introduction to Phosphatase
 The phosphatases include:
 Alkaline phosphatase (ALP)
 Acid phosphatase (ACP)
 Red cell phosphatase
Phosphatases catalyze the following:
 Organic phosphate monoester + water 
Alcohol + Phosphate ion
118

Alkaline Phosphatase: Biochemical Theory and
Metabolic Pathway
 Hydrolase enzyme catalyzes dephosphorylation
reactions:
 Removal of phosphate groups from nucleotides,
proteins, and alkaloids
 Alkaline pH improves activity.
 ALP has an optimum activity at pH of about 10.
119

Sources of Alkaline Phosphatase
Hepato-
cellular
Hepato-
biliary
Osteo-
blasts
(Bone)
Intest-
inal
Mucosa
Placenta
NA ALP ALP ALP ALP
It is present in serum, liver, bone, intestinal mucosa, placenta, renal tubule cells
and leucocytes.
The activity in normal serum is predominantly of liver and bone origin.
NA = not applies. ALP is not found in hepatocellular tissues but in heptobiliary
tissues.
ALT and AST are the enzymes commonly found in hepatocelluar (parenchymal)
tissues
120

Isoenzymes
Alkaline phosphatase has two main forms:
 Bone sources
 Intestinal and liver sources
121

Alkaline Phosphatase
Isoenzyme
Characteristics
Name of Isoenzyme Hepatic Bone
Heat Stability Stable at 560 C for 30
minutes
Labile: disappears
at 560 C within 10
minutes
Electrophoretic Order Most anodic Intermediate
Chemical Inhibition Moderate inhibition by
urea but low inhibition
by l-phenylalanine
Strong inhibition by
urea but low inhibition
by l-phenylalanine
122

Isoenzyme
Characteristics
Intestinal Placental Other
Intermediate labile:
disappears at 560 C
within 15 minutes
Stable at 560 C for 30
minutes
Regan isoenzyme:
most stable
Cathodic -bone
fraction
Migrates with hepatic
or bone forms
Renal isoenzyme: rare
but most cathodic
Strong inhibition by l-
phenylalanine.
Resistance to urea but
phenylalanine.
Regan isoenzyme:
phenylalanine.
123

Clinical Significance of ALP
Results
 High concentration of ALP in hepatobiliary cells
 Biliary inflammation or ductal obstruction
 Cellular inflammation and necrosis
 Increased with bone diseases of
osteoblastic activity
124

Cholestasis and ALP
 Release of ALP into the circulation.
 Cholestasis may cause ALP increased 3-10 X the
normal levels.
 Serum total and direct bilirubin are increased.
125

Test Methodology: Alkaline
Phosphatase
 Analysis by the Bessey Lowry and Brock ALP method
 p-nitrophenyl phosphate + H2O –(ALP, glycine buffer,
Mg2+, pH 10.5) p-nitrophenol + PO4 3+
-> yellow quininoid chromagen
measure increase in Abs. at 400 nm at 37 0 C
126

Test Methodology: Alkaline
Phosphatase
Analysis of alkaline phosphatase: Bowers and McComb
modified method:
 4-nitrophenyl phosphate + H2O –(ALP, Mg2+, pH 10.3)
4-nitrophenoxide
Increase in Abs. at 405 nm at 37 0 C
Photometer used for two-point analysis.
127

Per Minute
 In ALP the absorbance increases over time
 The result is A X Factor
Min
 Where F405 = positive number
128

Analysis Results Example
The following results for ALP were determined
Are the results progressing in the expected direction?
Time
(min)
Absorbance
0 1.250
1 1.350
2 1.449
3 1.551
Answer: yes, they are increasing
129

ALP analysis
What is the  Abs for each minute?
Answer: 0.100/min; 0.099/min; and 0.102 /min
Are the results consistent?
Answer: Yes, absorbance increases
consistently
Average  Abs = 0.100 /min
130

Per Minute from the
Example
 F = 2040
 Calculate the final activity in U/L for this test result.
Answer:  Abs = 0.100 /min x 2040 = 204
U/L
131

Calculation of Activity
 ALP is reported as U/L activity.
What does U/L mean?
Answer: Activity is the amount of enzyme able to
convert 1 micromole of substrate to product per minute
per liter. U/L.
132

Specimens for Alkaline
Phosphatase Analysis
 Non-hemolyzed serum
 heparinized plasma
 Fresh or refrigerated
133

Interpretation of Alkaline Phosphatase
Reference ranges vary with method used:
 53 -128 U/L
 2x or more increases in serum or plasma:
 Bone cancer, bone disease (such as Paget’s)
 Hepatobiliary disease such as cholestasis,
cholelithiasis
or gall stone
134

Quality Control
135

Methods
Pre-analytic Errors
 Anticoagulants that remove Ca or Mg
 Not Fresh Sample
 False increased activity over time due to increasing pH
of the sample
 Hemolysis:
 Poor sample collection
 Poor processing
136

Sources of Errors in
Analytic Errors
 Substances that absorb light at 405 nm:
 Lipids (lipemia)
 Bilirubin
 Hemoglobin
137

Sources of Errors
 Too Acidic
 Substrate exhaustion from excessively elevated enzyme
levels
 Unstable temperature
readings
138

Reporting and
Documentation
Results should be carefully review before reporting to
clinicians.
Documentation of occurrences patient and quality
control results in logbooks is necessary.
Avoid common Post-analytic Errors:
 Wrong name
 Incorrect units or reference range
 Transcribing wrong number
 Report too late
139

Problem-solving results
The following results for ALP were determined:
Are the results progressing in the expected direction?
Time (min) Absorbance
0 1.350
1 1.369
2 1.350
3 1.401
Answer: No, they are increasing and then decreasing
140

Problem-solving:
Fluctuating Results
What might cause unstable absorbance readings?
Answer: Unstable temperature or photometer
readings.
141

Problem-solving Results
A patient serum alkaline phosphatase result printed
from the analyzer as NL: nonlinear due to substrate
exhaustion. 20 microliters (uL) of serum is mixed
with 40 uL of diluent and the sample was analyzed
again. Results on the next screen.
142

Problem-solving
Results: Dilution for
ALP Analysis
The diluted result printed out as 550 U/L and a manual
calculation is required.
What is the actual ALP activity?
Answer: 3 x 550 = 1650 U/L
143

Cirrhosis and ALP
These results were obtained from a patient suspected of having cirrhosis,
causing chronic scarring of the liver and loss of liver function.
Describe what you observe regarding the liver enzymes.
Test Result Reference
T. Bilirubin 3.1 0.0-2.0 mg/dL
Dir. Bilirubin 0.5 0.0-0.2 mg/dL
ALT 65 <34 U/L
ALP 805 53 -128 U/L
Answer: ALP, the biliary enzyme is 8x the normal level and
ALT, the hepatocellular is almost 2x the normal level.
144

Sources of Acid Phosphatase
(ACP)
 All cells except RBCs
 Largest amounts in:
 Prostate gland (semen)
 Liver
 Spleen
 Breast milk
 Platelets
 Bone marrow
145

Acid Phosphatase: Biochemical Theory
and Metabolic Pathway
 Hydrolase enzyme catalyzes dephosphorylation of
phosphoric monoester
 Acid pH improves activity.
 Found in many tissues
 prostatic tissues and seminal fluid
 Non-prostatic sources
 Analysis is directed toward specific source of ACP eg.
prostatic ACP or non-prostatic ACP
146

Clinical Significance of ACP
 Prostatic diseases
 Metastatic prostatic cancer
 Forensic investigation of rape victims
 Bone disease
 Metastatic bone cancer
147

Two-point photometric
analysis of ACP
The Bessey-Lowry and Brock (BLB) method
 determination of total ACP
P-Nitrophenyl phosphate (PNPP) + H2O → p-
Nitrophenol + Phosphate ion (Colorless) -> (Yellow
chromagen) at 410nm
148

Two-point photometric
analysis of ACP
 Determination of prostatic ACP
 PNPP procedure repeated with tartarate solution to
measure only the 'tartarate-stable' or non-prostatic
enzyme activity.
 Prostatic ACP activity = Total ACT activity –
Nonprostatic ACP activity
149

Assay of Prostatic ACP
 Immunological Methods
 Radioimmunoassay (RIA)
 Chemiluminescence immunoassay
150

Specimens for ACP
 Serum
 Separated immediately from whole blood
 Add 5 mol/L acetic acid per mL of serum or sodium
citrate to preserve
 Store up to 1 week in refrigerator
 Vaginal Swab
 Forensic
151

Interpretation of Acid
Phosphatase:
 Serum or plasma reference ranges vary with method
 For example: 0.0 – 4.3 U/L male Total ACP
 Prostatic cancer causes 2x or more increases in serum
or plasma
 0-0.6 U/L male Prostatic ACP
152

Quality Control
153

Sources of Error in ACP
Hemolysis
 Serum not separated immediately from whole blood
 Nonacidified serum
 Lipemia
 Serum > 1 week in refrigerator
 Anticoagulants (other than citrate)
154

Learning Objectives
and physicochemical properties of creatine kinase (CK)
CK and CK isoenzymes
 Describe the principles of CK and CK isoenzyme analysis in
156

Learning Objectives
and postanalytical errors in CK and CK isoenzyme analysis.
 Interpret results of CK and CK isoenzymes compared to
reference ranges.
157

Outline of Lecture: CK and CK
Isoenzymes
 Introduction
 Source
 Specimen
 Quality Control
 Summary
158

Introduction to Creatine Kinase
 Creatine N-phosphotransferase; (CK)
 Transferase
(CK, pH 9.0, Mg++)
Creatine + ATP   Creatine phosphate+ ADP
(CK, pH 6.7, Mg++)
Unstable enzyme: temperature and ions affect
159

Sources of Creatine Kinase
 Skeletal Muscle
 Brain
 Cardiac Muscle
 Some others
 Kidney
 Lung
160

Isoenzymes of CK
 Dimer of M or B polypeptides
 M= muscle
 B = brain
 CK-1 (BB)
 CK-2 (MB)
 CK-3 (MM)
 Possible permutation of the subunits gives rise to three
distinct isoenzymes,
 Arranged in decreasing electrophoretic anodal mobility:
CK-1 (BB), CK-2 (MB), and CK-3 (MM).
161

Clinical Significance of Creatine
Kinase
 Diseases of
 Skeletal muscle
 Muscular Dystrophy
 Cardiac muscle
 Acute Myocardial Infarction
 Brain
 CNS disease
 Stroke/ cerebral vascular accident
 Other: thyroid disease
162

Time Frame of CK elevation
with Myocardial Infarction
 Elevates within a few hours of AMI
 Returns to normal by 48 hours
163

Methods of Analysis of CK
 Continuous Monitoring Kinetic method
 Coupled enzyme
 Colorimetric/ photometric
 Fluorometric
164

Principle of CK Methods:
Continuous Monitoring
Creatine phosphate + ADP –CK, pH 6.7
Creatine + ATP
ATP + glucose –HK glucose-6-P + ADP
glucose-6-P + NADP+  6 phosphogluconate + NADPH
+ H+
Measured photometrically (continuous monitoring)
Absorbance should increase.
165

Principles of CK Isoenzyme
Methods
 Electrophoresis:
 Samples are separated by electrical charge on agarose
or cellulose membrane.
 CK assay substrate is incubated on membrane.
 NADPH forms to indicate CK isoenzyme bands.
 Visualized with fluorescent densitometer at 360 nm
 Alternate method produces formazan bands visualized
with visible light densitometer.
166

Principles of CK Isoenzyme
Electrophoresis
 CK1 (BB) is fastest
 CK2 (MB) is
intermediate
 CK3 (MM) is slowest
167

Other CK Isoenzyme Methods
 Ion Exchange Chromatography
 Immunological Methods
 Immunoprecipitation
 Immunoassay
168

Specimen for CK Analysis
 Non-hemolyzed Serum
 Storage in refrigerator or freezer if not analyzed
promptly or as soon as
169

Reference Ranges and
Interpretation
 Total CK activity:
 Adult male: 15-105 U/L
 Adult female: 10-80 U/L
 CK1: 0%
 CK-2: < 4%-6% of Total CK
 CK-3: 94-100% of Total CK
 Reference ranges vary according to age of patient and
method used
170

Sources of Error in CK Analysis
 Sample not fresh or exposed to heat
 Anticoagulants such as citrate or fluoride
 Gross hemolysis causes analytic error
171

Learning Objectives
and physicochemical properties of Lactate dehydrogenase
(LD)
LD and LD isoenzymes
 Describe the principles of LD and LD isoenzyme analysis in
173

Learning Objectives
and postanalytical errors in LD and LD isoenzyme analysis.
 Interpret results of LD and LD isoenzymes compared to
reference ranges.
174

Outline of Lecture: LD and LD
Isoenzymes
 Introduction
 Source
 Specimen
 Quality Control
 Summary
175

Introduction to Lactate
Dehydrogenase
 L-lactate: NAD+ oxidoreductase, LD
 Oxidase
(pH 8.8-9.8)
 L-Lactate + NAD+ → Pyruvate + NADH + H+
( pH 7.4-7.8)
Enzyme specificity includes other a hydroxy acids
LD is inhibited by mercuric ions
176

Sources of LD
 All cells
 Heart
 Liver
 Kidney
 Erythrocytes
 Skeletal Muscle
 Brain
177

Clinical Significance of LD
 Myocardial infarction
 Liver disease (hepatic inflammation)
 Hemolytic conditions
 Malignancies
 Skeletal muscle disease
 Renal disease
 Pulmonary embolisms
178

Clinical Significance of LD…'rich
man's ESR
 Time Frame of LD following Acute Myocardial
Infarction (AMI)
 Elevated 2-3 days after AMI
 Returns to normal by 7-10 days after AMI
179

Analytical Methodology of Total
Lactate Dehydrogenase Activity
Reverse method (P L)
NADH + H+ + Pyruvate -- LDH  Lactate + NAD+
 Absorbance of NAD can be measured with photometer
at 340 nm
 The molar absorptivity (epsilon) of NAD at 340 nm is
6220 cm. L/ mole
180

Analytical Methodology of Total
Lactate Dehydrogenase Activity
 End-point colorimetric method
 Test principle: Pyruvate released by LDH is reacted
with 2, 4-dinitrophenyl hydrazine to form the
corresponding golden-brown colored hydrazone at an
alkaline pH. The intensity of the color is proportional
to enzyme activity and is measured at 410 nm.
181

Specimen for LD
 Stored at room temperature
182

Interpretation of LD Results
 Age Specific Reference Ranges
 Dependent on methods
 Serum for Adult: 100-190 U/L
 CSF for Adult: 10% of serum value
 Compare patient result to reference range to assess for
cardiac, liver, skeletal muscle or other diseases.
183

Quality Control
184

Sources of Error in LD
with diluted sample
 Loss of activity if frozen or stored more than 3 days at
room temperature
 Use of anticoagulant is source of error
185

Documentation of LD
Enzyme Analysis
186

Isoenzymes
 Multiple forms of one type of enzyme
 React with the same substrate
 Composed of slightly different polypeptide chains
 Have some unique characteristics such as temperature
inactivation or clinical significance
 LD1 or HHHH
 LD 5 or MMMM
188

5 LD Isoenzymes
 HHHH, LD1: cardiac muscle, erythrocytes, brain, and
renal cortex
 HHHM LD2: cardiac muscle, erythrocytes, brain and
renal cortex
 HHMM LD3: lung, spleen and the platelets
 HMMM LD4: liver and skeletal muscle
 MMMM LD5: liver and skeletal muscle
189

Clinical Significance of LD
Isoenzymes
 LDH-1 and LDH-2 :cardiac muscle, erythrocytes, and
renal cortex
 LDH-3: lung, spleen and the platelets
 LDH-4 and LDH-5: liver and skeletal muscle.
 Diseases affecting these organs and tissues will cause
elevation of individual isoenzyme % compared to
reference ranges.
190

Method of Separation of LD
Isoenzymes: Electrophoresis
 pH 8.0 buffer
 migrated with electrical current
 agarose or cellulose membrane.
 d,l- lactate + NAD + Substrate is placed on separated
fractions, incubated at 37C to develop colored
formazen bands.
191

Method of Separation of LD
Isoenzymes: Electrophoresis
 Densitometric Scan of Normal Serum
 Note the anode view is on the right side.
192

Calculation of Results of LD
isoenzyme electrophoresis
 Densitometer is used to determine isoenzyme %
 % OD increases with larger, darker bands.
 Total % must add up to 100%
 The electrophoretic pattern is also significant.
193

Interpretation of LD Isoenzyme
Electrophoresis Results
 Lane A: myocardial
infarction
 Lane B: normal
 Lane C: liver disease
 1 = LD1 etc.
194

Other Methods for Isoenzyme
of LD
 Selective Chemical Inhibition
 Ion exchange chromatography
 Immunoprecipitation
 Selective Substrate to measure 2 hydrobutryase
activity
195

Interpretation of LD Isoenzyme
Results
 LD1: 14- 26%
 LD2: 29-39%
 LD3: 22-26%
 LD4: 8-16%
 LD5: 6-16%
 Compare patient results to reference ranges to indicate
if diseases of heart, liver or others may be present.
Isoenzyme patterns provide additional information.
196

Specimen for LD isoenzymes
 Stored at room temperature
197

Sources of Error in LD
Isoenzymes
 LD 1 and LD 2
 Loss of activity if frozen or stored more than 3 days at
room temperature
 LD 4 and LD5
 Use of anticoagulant is source of error
198

Documentation of LD
Isoenzyme Enzyme
Analysis
199

Summary LD and LD
Isoenzymes
 Discussion of source and clinical Significance of LD and
LD isoenzymes
 Description of methods of analysis, specimen,
interpretation of results, QC, sources of error and reporting
and documentation procedures for LD and LD isoenzymes
200

Lecture Objectives
Upon completion of this lecture, the student will be
able to:
1. Describe the anatomy of the pancreas
2. Define pancreatitis and distinguish between acute
and chronic forms of the disease
3. Discuss methodology and principles of analysis used
in amylase and lipase measurement
203

Lecture Objectives
able to:
4. Compare and contrast the advantages and
disadvantages of the following amylase methods
used to evaluate pancreatic function: a. Amyloclastic
b. Saccharogenic;
c. Chromolytic;
d. Other
204

Lecture Objectives
able to:
5. Discuss amylase and lipase values and activity in the
diagnosis of pancreatic disease and other conditions
that may cause elevation of these enzymes
6. Discuss the amylase/creatinine clearance ratio
(ACCR) and its usefulness as a diagnostic tool
205

Outline of Lecture: Amylase
and Lipase
 Introduction
 Source
 Specimen
 Quality Control
 Summary
206

Introduction to Amylase
 Nomenclature: (EC 3.2.1.1; 1,4-a-D-Glucan
Glucanohydrolase; AMS)
 Group of hydrolases
 Split large polysaccharide (starches) to a-D-glucose
 2 types
 Alpha (endo-amylase): human form
 Beta (exo-amylase): plant and bacterial origin;
207

Amylase: Source
 Produced by the pancreas
 Also produced by other
organs, particularly the salivary
glands
208

Function of Amylase
 Secreted through the pancreatic duct into the duodenum,
where it helps break down dietary carbohydrates
 Part of digestion
 Amylases are secreted by the salivary and pancreatic
glands into their respective juices, which enter the
gastrointestinal tract.
 These enzymes are important for the digestion of
ingested starches,
 but the amylase from the pancreas plays the major role,
since the salivary amylase soon becomes inactive in the
acid condition prevailing in the stomach.
209

Urinary Amylase
 Normal amylase is filtered into urine
 Elevated urinary amylase in chronic pancreatitis
 Macroamylassemia
 Rare Ig complexed amylase that is too large to filter into
urine
 Not clinically significant
210

Clinical Significance of Amylase
Test
 Why the test is performed
 This test is performed to evaluate pancreas function,
specifically for acute and chronic pancreatitis
 Not entirely specific to pancreas
 Mumps
211

Clinical Significance of Amylase
 Pancreatitis
 Inflammation of the pancreas
 Two types: acute and chronic
 A serious condition most commonly caused by either alcohol
toxicity or gallstones
212

Symptoms of Acute
Pancreatitis
 Severe abdominal pain
 Swollen tender abdomen
 Nausea
 Vomiting
 Fever
 Sweating
 Rapid pulse
 Jaundice
213

Chronic Pancreatitis
 Symptoms similar to acute
 Damage to pancreas and declining function
 First attack alcohol related
 Pancreatic calcification years after first clinical
presentation
 Chronic pancreatitis can develop after one acute attack
if the ducts become damaged
 Middle age men
214

List of Amylase Analytic
Methods
 Viscosimetric Method (Historical)
 Iodometric (Amyloclastic) Method
 Saccharogenic Method
 Chromolytic Method
 New automated Methods
215

Amylase Methods of Analysis
Saccharogenic / Somogyi Method:
 Starch  Reducing sugars (maltose, glucose residues)
 Reducing sugars + Cu++  Oxidized sugars + Cu+
 Cu+ + Phosphomolybdic acid 
Phosphomolybdous acid + Cu++
[Abs. Blue colored product measured with photonmeter
at 680 nm]
216

 Saccharogenic/Amylometric Caraway Procedure
 Starch  Maltose and other fragments
 Unhydrolyzed Starch +Iodine Starch-Iodine
complex
 Absorbance of Violet blue-black product measured
with photometer at 660 nm
217

Chromolytic Method
 Amylose-Dye Substrate –(presence of amylase)-
chromagen
 Chromagen is measured with Automated analyzer
method- common
218

Specimen Requirements for
Amylase
 Blood Specimen requirements
 Non-haemolyzed serum or heparinized plasma
 Urine
 Random or timed urine (2-hour) specimens
219

Interpretation of Amylase
Results
Compare the Patient Result with the Reference
Range
 Reference Ranges
 Serum: 70 - 340 IU/L
 Urine: up to 300 IU/L per hour
 values may vary lab to lab
 Elevated amylase levels correlate with pancreatitis
220

Sources of Errors for Amylase
Methods
 Interferences
 Inhibitors in the reaction due to :
 Wrong pH
 Lack of obligate activators (Ca, Cl and Br)
221

Introduction to Lipase
 Water-soluble enzyme
 Catalyzes hydrolysis of
ester bonds in water
 Esterase
 Lipid Substrate
 Pancreatic lipase is
secreted as the active
enzyme
 Secreted from pancreatic
duct into duodenum
 Concentration in serum is
very low
222

Clinical Significance of Lipase
 Elevated levels may indicate:
 Cholecystitis (with effects on the pancreas)
 Pancreatic cancer
 Pancreatitis
 Stomach ulcer or blockage
 Viral gastroenteritis
 Special considerations
 Drugs that may alter test results include bethanechol,
cholinergic medications, codeine, indomethacin,
meperidine, methacholine, and morphine
223

Methods and Principles of
Lipase Analysis
 Titration of released fatty acids (Cherry-Crandall
method)
 Triglyceride – LPS Monoglyceride + 2 fatty acids
 Fatty acid + NaOH titration to neutrality using
phenolphthalein indicator
 Results are determined from volume of base added
224

Lipase Method
Emulsion clearing
 Turbidimetric or nephelometric monitoring of
decrease in size of emulsion of substrate after
action of lipase
 Light scatter is measured
 Widely used in automated
spectrophotometric or nephelometric analyzer
225

Other Lipase Methods
 Colorimetric (not common)
 Reaction A: Triglyceride – LPS Monoglyceride + 2 fatty
acids
 Fatty acids react with Spectru ® Cationic Blue dye 
blue complex measured with colorimeter
 Coupled Enzymatic
 Lipase acts on substrate glycerol (quantified by
enzymatic reaction)
 Used on automated dry slide chemistry instrument
226

Specimen Requirements for
Lipase
 Serum;
 Storage at room temperature for <1 week
 Storage for < 3 weeks in refrigerator
 Stool/ duodenal fluid
227

Interpretation of Lipase Results
Compare the Patient Result with the Reference Range
 Reference Range
 Serum Reference Ranges:
 0 - 62 U/L. Normal values may vary lab to lab
228

Quality Control for Lipase
Methods
be analyzed along with patient samples, using quality
control rules for acceptance or rejection of the
analytical run.
229

Sources of Error for Lipase
 Bacterial contamination of specimen
 Patients with rheumatoid arthritis may produce
nonlinearity in kinetic assay of lipase
 Reagents limit false positive interference
230

Lipase Summary
 In acute pancreatitis, elevated lipase levels usually
parallel blood amylase concentrations, although
amylase levels tend to rise and fall a bit sooner than
lipase levels
 Drugs that may increase lipase levels include codeine,
indomethacin, and morphine
231

Amylase/Creatinine Clearance
Ratio ACCR
 This test is used to differentiate between pancreatitis
and other causes for elevated amylase in serum
compared to urine.
 Details about the source and physiology of creatinine
were discussed in your renal function chapter.
232

Principle of Methods
Ratio ACCR
 Refer to principle of methods for Amylase and
Creatinine
233

Specimen Amylase/Creatinine
Clearance Ratio ACCR
 Specimens required:
 Random or short-term urine specimen (2-hour) for
amylase and creatinine assay
 Serum specimen for amylase and creatinine assay
234

Ratio ACCR
 The ACCR is a useful diagnostic test to differentiate
clinical diagnosis
ACCR =
urine amylase (U/L) x Serum Creatinine (mg/L) x 100
serum amylase (U/L) x urine creatinine (mg/L)
235

Interpretation of ACCR
 Reference Range: 2-5%
 Increased value in pancreatitis
 Other causes of increase ACCR
 Decreased value in Macroamylasemia.
236

Summary of Lesson
This lesson included:
 Definition of pancreatitis and how to distinguish
between acute and chronic forms of the disease
 Methodology and principles of analysis used in
amylase and lipase measurement
237

Summary of Lesson
 Amylase and lipase values and the diagnosis of pancreatic
disease and other conditions that may cause elevation of
these enzymes
 Appropriate specimen(s) for amylase and lipase
measurement and pre-analytic variables
 Review / performance of either manual, semi-automated,
or automated analysis of amylase and lipase
238

References
 Burtis, Carl A., and Ashwood, Edward R.. Tietz: Fundamentals of Clinical
Chemistry. Philadelphia, 2001
 http://www.arizonatransplant.com/images/pancreas_large_2.JPG
 http://www.cellscience.com/Reviews5/Nunemaker1.jpg
 http://www.labtestsonline.org/understanding/analytes/amylase/test.html
 http://www.montana.edu/wwwai/imsd/alcohol/Vanessa/vwpancreas.htm
 http://www.nlm.nih.gov/MEDLINEPLUS/ency/presentations/100149_2.htm
 http://www.principalhealthnews.com/topic/adam1003465
 http://www.webmd.com/digestive-disorders/amylase-17444
 Kaplan, L.A., and Pesce, A. J.. Clinical Chemistry, Theory, Analysis, and
Correlation. St. Louis, 1989
 Wu, Alan. Tietz Clinical Guide to Laboratory Tests. St. Louis, 1995
 Arneson, W and J Brickell: Clinical Chemistry: A Laboratory Perspective 1st ed.
FA Davis, Philadephia 2007
239

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