Methods for the determination in serum and urine

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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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Examples Type Group No.
i) lactate dehydogenase ii) glucose oxidase iii) tyrosinase Oxidoreductase i) anaerobic dehydrogenase ii) aerobic oxidase iii) aerobic dehydrogenase 1
Alanine amino transfesrase (ALT) Aspartate amino transferase (AST) Creatine Kinase (CK) Transferases 2
Lipase Cholinesterase Hydolases 3
Pyruvate decarboxylase Lyases 4
Triphosphate isomerase Isomerase 5
Acetyl CoA synthetase Ligases 6
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Types of enzymes

• Enzyme Commision (EC) International enzyme
  numbering system
• 4 figures separated by dotes e.g. 1.1.2.1
• Explanation
• 1. 1. 2.
  1.
• Group no group acted on subgroup acted on
  the individual enzyme

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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
• 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 CK
• Catalyzes the reaction ATP creatine
  ADP creatine phosphate
• CK isoenzymes and clinical importance
• Isoenzyme tissue or organ present
• CK-BB (CK-1) brain 98
• CK-MB (CK-2) heart muscle 20
• CK-MM (CK-3) muscle 96

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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
• Creatine phosphate ADP
  ATP creatine
• hexokinase
• ATP glucose ADP
  Glucose-6-phosphate
• G-6-P Dehydrogenase
• 3. G-6-P NADP
  6-phosphogluconate NADPH H

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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 Pyruvate
  NADH H

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2) Lactate dehydrogenase (LD) (EC 1.1.1.27)

• Iso-enzymes of LD and clinical importance

Clinical significant Site in circulation Subunit LD iso-enzyme
MI Haemolytic anaemia Haemolyzed sample Acute renal failure Heart RBC Renal cortex 14-26 29-39 HHHH HHHM LD1 LD2
Pulmonary pneumonia Lymphocytosis Acute pancreatitis Lung Lymphocyte Pancrease 20-26 HHMM LD3
Hepatic carcinoma Hepatic necrosis Skeletal muscle injury Liver Skeletal muscle 8-16 8-16 HMMM MMMM LD4 LD5
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2) Lactate dehydrogenase (LD) (EC 1.1.1.27)

• In normal adult circulation LD2 gt LD1, MI LD1 gt
  LD2 and the LD1 gt LD2 ratio is gt1. 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