Transcript CLINICAL CHEMIISTRY (MT 305) CARBOHYDRATE LECTURE ONE

Methods for the determination
in serum and urine
Dr. Essam H. Aljiffri
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Types of enzymes
- The International Union of Biochemistry (IUB) in
1964 has suggested that enzymes are arranged
in groups according to their functional catalytic
activities.
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Group
No.
1
Type
Oxidoreductase
i) anaerobic dehydrogenase
ii) aerobic oxidase
iii) aerobic dehydrogenase
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Transferases
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Examples
i) lactate dehydogenase
ii) glucose oxidase
iii) tyrosinase
Alanine amino transfesrase (ALT)
Aspartate amino transferase (AST)
Creatine Kinase (CK)
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Hydolases
Lipase
Cholinesterase
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Lyases
Pyruvate decarboxylase
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Isomerase
Triphosphate isomerase
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Ligases
Acetyl CoA synthetase
Types of enzymes
Enzyme Commision (EC) International enzyme numbering system
4 figures separated by dotes e.g. 1.1.2.1
Explanation
1.
Group no:
4
1.
2.
1.
group acted on: subgroup acted on: the individual enzyme
General consideration in enzyme
assays in the clinical laboratory
• In enzyme assays the activity of the enzyme and
not the enzyme concentration is measured
• Clinically important enzymes, i.e. Enzymes of
diagnostic value, are those whose activities are
reflective of the condition of a certain function in the
body or an organ and the determination of their
activity will assist in the diagnosis or the
management of diseases in the patient.
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General consideration in enzyme
assays in the clinical laboratory
• Serum is the preferred specimen type for enzyme assays
• Avoid haemolysis;
– RBCs contain high concentration of some enzymes such
as LDH, transferases and G6PD
– Haemoglobin may interfere with some assays especially
those which include color production
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General consideration in enzyme
assays in the clinical laboratory
• Never shake the serum or the reaction mixture
vigorously as this may denature the enzyme, mix
the serum and reagent gently.
• Avoid using NaF/ K oxalate tube as NaF is a
enzyme inhibitor
• Check if the patient is taking drugs that effect
enzyme activity
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General consideration in enzyme
assays in the clinical laboratory
• Avoid prolonged application of tourniquet as it effect
some enzymes such as LDH
• Note the physical condition of the patient (e.g.
exercise or long walk may effect CK activity)
• Some enzymes are sex related i.e. higher or
present in one sex type (e.g. prostatic ACP)
• Some enzymes are age related (e.g. ALP)
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General consideration in enzyme
assays in the clinical laboratory
• Some enzymes catalyze both direction of the
reaction while others catalyze one direction only
• Many enzymes exit as isoenzymes (different forms
in different organs), such enzymes have a good
diagnostic value, the isoenzyme related to the organ
should be analyzed together with the total enzyme
activity; e.g. CK & CK-MB
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General consideration in enzyme
assays in the clinical laboratory
Because the enzyme activity is measured, many conditions
affect such reactions and include;
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Substrate type and concentration
Product type and concentration
Amount of enzyme present
Buffer type and pH
Activators and Coenzymes
Temperature of the reaction
Specificity of the enzyme to substrate
Presence of inhibitors
Direction of reaction (forward or reverse direction)
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General consideration in enzyme
assays in the clinical laboratory
• Due to the effects of various conditions on enzyme
activity, each lab must determine its normal range
for the enzymes in question and not rely on
published data.
• The effect of the various conditions on enzyme
activity make enzyme assays less precise than
other smaller analytes so a coefficient of variation
(cv) of up to 10% is acceptable.
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General consideration in enzyme
assays in the clinical laboratory
• Kinetic enzyme (rate of reaction ) assays are to be
used instead of end point (two-point) assays
because they provided better accuracy.
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Some enzymes of diagnostic importance.
1) Creatine kinase (creatine phosphokinase) (CK) (EC: 2.7.3.2)
Activity:
Catalyzes the reaction ATP + creatine
CK
ADP + creatine phosphate
CK isoenzymes and clinical importance:
Isoenzyme
CK-BB (CK-1)
CK-MB (CK-2)
CK-MM (CK-3)
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tissue or organ
brain
heart muscle
muscle
% present
98 %
20 %
96 %
1) Creatine kinase (creatine phosphokinase)
(CK) (EC: 2.7.3.2)
Method of analysis
Continuous monitoring (kinetic) method
(Scandinavian Committee on Enzyme, 1979)
(Association of Clinical Biochemists, UK, 1980)
Specimen
• Fasting serum (preferred)
• 50µmol/L N-acetylcysteine is added to serum immediately after
separation (activator for CK)
• Storage at 40C for up to 2 days
• Storage at – 200C for up to 30 days
• Avoid haemolysis
• Never repeat thaw-freeze
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1) Creatine kinase (creatine phosphokinase)
(CK) (EC: 2.7.3.2)
Principle
This is a rate kinetic method based on the reverse
reaction of the enzyme and coupled to other
enzyme reactions.
CK
1.
Creatine phosphate + ADP
ATP + creatine
hexokinase
2.
ATP + glucose
ADP + Glucose-6-phosphate
G-6-P Dehydrogenase
3.
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G-6-P + NADP+
6-phosphogluconate + NADPH + H+
1) Creatine kinase (creatine phosphokinase)
(CK) (EC: 2.7.3.2)
Principle
The rate of formation of ATP is monitored using
the increase in absorbance at 340 nm of NADPH
formed by the coupled reactions.
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Some enzymes of diagnostic importance.
2) Lactate dehydrogenase (LD) (EC 1.1.1.27)
This is a universal enzyme occurring in almost all tissues of
the body with higher concentration in cardiac muscle,
skeletal muscle, liver, kidney & rbc
Activity: Catalyzes the reaction:
LD
Lactate + NAD+
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Pyruvate + NADH + H+
2) Lactate dehydrogenase (LD) (EC 1.1.1.27)
Iso-enzymes of LD and clinical importance
LD iso-enzyme
Subunit
%
in circulation
LD1
LD2
HHHH
HHHM
14-26 %
29-39 %
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Site
Heart
RBC
Clinical
significant
Renal cortex
MI
Haemolytic anaemia
Haemolyzed sample
Acute renal failure
LD3
HHMM
20-26 %
Lung
Lymphocyte
Pancrease
Pulmonary pneumonia
Lymphocytosis
Acute pancreatitis
LD4
HMMM
8-16 %
Liver
LD5
MMMM
8-16 %
Skeletal
muscle
Hepatic carcinoma
Hepatic necrosis
Skeletal muscle injury
2) Lactate dehydrogenase (LD) (EC 1.1.1.27)
• In normal adult circulation LD2 > LD1, MI LD1 > LD2 and
the LD1 > LD2 ratio is >1. This called “flipped LD” pattern.
Method of analysis
Continuous monitoring (kinetic) method
(Scandinavian Committee on Enzymes, 1974)
Specimen
• Fasting serum (preferred)
• Avoid haemolysis
• Never repeat thaw-freeze
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2) Lactate dehydrogenase (LD) (EC 1.1.1.27)
Principle
• This is a kinetic method based on the reverse reaction of
the enzyme
LD
• Pyruvae + NADH + H+
Lactate + NAD+
• The rate of reaction is monitored as pyruvate is
converted to lactate by observing the decrease in
absorbance at 340 nm as NADH is oxidized to NAD+
• This is faster than the forward reaction
• Less expensive than the forward reaction
• It requires less concentration of reagents
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